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

  • Tammy L Romanuik1,

    Affiliated with

    • Gang Wang1,

      Affiliated with

      • Olena Morozova1,

        Affiliated with

        • Allen Delaney1,

          Affiliated with

          • Marco A Marra1 and

            Affiliated with

            • Marianne D Sadar1Email author

              Affiliated with

              BMC Medical Genomics20103:43

              DOI: 10.1186/1755-8794-3-43

              Received: 20 April 2010

              Accepted: 24 September 2010

              Published: 24 September 2010

              Abstract

              Background

              There is no cure for castration-recurrent prostate cancer (CRPC) and the mechanisms underlying this stage of the disease are unknown.

              Methods

              We analyzed the transcriptome of human LNCaP prostate cancer cells as they progress to CRPC in vivo 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.

              Results

              Three million tags were sequenced using in vivo 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 (CCNH, CUEDC2, FLNA, PSMA7), steroid synthesis and metabolism (DHCR24, DHRS7, ELOVL5, HSD17B4, OPRK1), neuroendocrine (ENO2, MAOA, OPRK1, S100A10, TRPM8), and proliferation (GAS5, GNB2L1, MT-ND3, NKX3-1, PCGEM1, PTGFR, STEAP1, TMEM30A), but neither supported nor discounted a role for cell survival genes.

              Conclusions

              The in vivo gene expression atlas for LNCaP was sequenced and support a role for the androgen receptor in CRPC.

              Background

              Systemic androgen-deprivation therapy by orchiectomy or agonists of gonadotropic releasing hormone are routinely used to treat men with metastatic prostate cancer to reduce tumor burden and pain. This therapy is based on the dependency of prostate cells for androgens to grow and survive. The inability of androgen-deprivation therapy to completely and effectively eliminate all metastatic prostate cancer cell populations is manifested by a predictable and inevitable relapse, referred to as castration-recurrent prostate cancer (CRPC). CRPC is the end stage of the disease and fatal to the patient within 16-18 months of onset.

              The mechanisms underlying progression to CRPC are unknown. However, there are several models to explain its development. One such model indicates the involvement of the androgen signaling pathway[14]. Key to this pathway is the androgen receptor (AR) which is a steroid hormone receptor and transcription factor. Mechanisms of progression to CRPC that involve or utilize the androgen signaling pathway include: hypersensitivity due to AR gene amplification [5, 6]; changes in AR co-regulators such as nuclear receptor coactivators (NCOA1 and NCOA2) [7, 8]; intraprostatic de novo synthesis of androgen[9] or metabolism of AR ligands from residual adrenal androgens[10, 11]; AR promiscuity of ligand specificity due to mutations[12]; and ligand-independent activation of AR by growth factors [protein kinase A (PKA), interleukin 6 (IL6), and epidermal growth factor (EGF)][1315]. Activation of the AR can be determined by assaying for the expression of target genes such as prostate-specific antigen (PSA)[16]. Other models of CRPC include the neuroendocrine differentiation [17], the stem cell model [18] and the imbalance between cell growth and cell death [3]. It is conceivable that these models may not mutual exclusive. For example altered AR activity may impact cell survival and proliferation.

              Here, we describe long serial analysis of gene expression (LongSAGE) libraries[19, 20] made from RNA sampled from biological replicates of the in vivo LNCaP Hollow Fiber model of prostate cancer as it progresses to the castration-recurrent stage. Gene expression signatures that were consistent among the replicate libraries were applied to the current models of CRPC.

              Methods

              In vivo LNCaP Hollow Fiber model

              The LNCaP Hollow Fiber model of prostate cancer was performed as described previously[2123]. All animal experiments were performed according to a protocol approved by the Committee on Animal Care of the University of British Columbia. Serum PSA levels were determined by enzymatic immunoassay kit (Abbott Laboratories, Abbott Park, IL, USA). Fibers were removed on three separate occasions representing different stages of hormonal progression that were androgen-sensitive (AS), responsive to androgen-deprivation (RAD), and castration-recurrent (CR). Samples were retrieved immediately prior to castration (AS), as well as 10 (RAD) and 72 days (CR) post-surgical castration.

              RNA sample generation, processing, and quality control

              Total RNA was isolated immediately from cells harvested from the in vivo Hollow Fiber model using TRIZOL Reagent (Invitrogen) following the manufacturer's instructions. Genomic DNA was removed from RNA samples with DNaseI (Invitrogen). RNA quality and quantity were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada) and RNA 6000 Nano LabChip kit (Caliper Technologies, Hopkinton, MA, USA).

              Quantitative real-time polymerase chain reaction

              Oligo-d(T)-primed total RNAs (0.5 μg per sample) were reverse-transcribed with SuperScript III (Invitrogen Life Technologies, Carlsbad, CA, USA). An appropriate dilution of cDNA and gene-specific primers were combined with SYBR Green Supermix (Invitrogen) and amplified in ABI 7900 real-time PCR machine (Applied Biosystems, Foster City, CA, USA). All qPCR reactions were performed in triplicate. The threshold cycle number (Ct) and expression values with standard deviations were calculated in Excel. Primer sequences for real-time PCRs are: KLK3, F': 5'-CCAAGTTCATGCTGTGTGCT-3' and R:' 5'-CCCATGACGTGATACCTTGA-3'; glyceraldehyde-3-phosphate (GAPDH), F': 5'-CTGACTTCAACAGCGACACC-3' and R:' 5'-TGCTGTAGCCAAATTCGTTG-3'). Real-time amplification was performed with initial denaturation at 95°C for 2 min, followed by 40 cycles of two-step amplification (95°C for 15 sec, 55°C for 30 sec).

              LongSAGE library production and sequencing

              RNA from the hollow fibers of three mice (biological replicates) representing different stages of prostate cancer progression (AS, RAD, and CR) were used to make a total of nine LongSAGE libraries. LongSAGE libraries were constructed and sequenced at the Genome Sciences Centre, British Columbia Cancer Agency. Five micrograms of starting total RNA was used in conjunction with the Invitrogen I-SAGE Long kit and protocol with alterations [24]. Raw LongSAGE data are available at Gene Expression Omnibus [25] as series accession number GSE18402. Individual sample accession numbers are as follows: S1885, GSM458902; S1886, GSM458903; S1887, GSM458904; S1888, GSM458905; S1889, GSM458906; S1890, GSM458907; S1891, GSM458908; S1892, GSM458909; and S1893, GSM458910.

              Gene expression analysis

              LongSAGE expression data was analyzed with DiscoverySpace 4.01 software [26]. Sequence data were filtered for bad tags (tags with one N-base call) and linker-derived tags (artifact tags). Only LongSAGE tags with a sequence quality factor (QF) greater than 95% were included in analysis. The phylogenetic tree was constructed with a distance metric of 1-r (where "r" equals the Pearson correlation coefficient). Correlations were computed (including tag counts of zero) using the Regress program of the Stat package written by Ron Perlman, and the tree was optimized using the Fitch program[27] in the Phylip package[28]. Graphics were produced from the tree files using the program TreeView[29]. Tag clustering analysis was performed using the Poisson distribution-based K-means clustering algorithm. The K-means algorithm clusters tags based on count into 'K' partitions, with the minimum intracluster variance. PoissonC was developed specifically for the analysis of SAGE data [30]. The java implementation of the algorithm was kindly provided by Dr. Li Cai (Rutgers University, NJ, USA). An optimal value for K (K = 10) was determined [31].

              Principle component analysis

              Principle component analysis was performed using GeneSpring™ software version 7.2 (Silicon Genetics, CA). Affymetrix datasets of clinical prostate cancer and normal tissue were downloaded from Gene Expression Omnibus [25] (accession numbers: GDS1439 and GDS1390) and analyzed in GeneSpring™. Of the 96 novel CR-associated genes, 76 genes had corresponding Affymetrix probe sets. These probe sets were applied as the gene signature in this analysis. Principle component (PC) scores were calculated according to the standard correlation between each condition vector and each principle component vector.

              Results

              LongSAGE library and tag clustering

              RNA isolated from the LNCaP Hollow Fiber model was obtained from at least three different mice (13N, 15N, and 13R; biological replicates) at three stages of cancer progression that were androgen-sensitive (AS), responsive to androgen-deprivation (RAD), and castration-recurrent (CR). To confirm that the samples represented unique disease-states, we determined the levels of KLK3 mRNA, a biomarker that correlates with progression, using quantitative real time-polymerase chain reaction (qRT-PCR). As expected, KLK3 mRNA levels dropped in the stage of cancer progression that was RAD versus AS (58%, 49%, and 37%), and rose in the stage of cancer progression that was CR versus RAD (229%, 349%, and 264%) for mice 13R, 15N, and 13N, respectively (Additional file 1). Therefore, we constructed nine LongSAGE libraries, one for each stage and replicate.

              LongSAGE libraries were sequenced to 310,072 - 339,864 tags each, with a combined total of 2,931,124 tags, and filtered to leave only useful tags for analysis (Table 1). First, bad tags were removed because they contain at least one N-base call in the LongSAGE tag sequence. The sequencing of the LongSAGE libraries was base called using PHRED software. Tag sequence-quality factor (QF) and probability was calculated to ascertain which tags contain erroneous base-calls. The second line of filtering removed LongSAGE tags with probabilities less than 0.95 (QF < 95%). Linkers were introduced into SAGE libraries as known sequences utilized to amplify ditags prior to concatenation. At a low frequency, linkers ligate to themselves creating linker-derived tags (LDTs). These LDTs do not represent transcripts and were removed from the LongSAGE libraries. A total of 2,305,589 useful tags represented by 263,197 tag types remained after filtering. Data analysis was carried out on this filtered data.
              Table 1

              Composition of LongSAGE libraries

              Library

              S1885

              S1886

              S1887

              S1888

              S1889

              S1890

              S1891

              S1892

              S1893

              Mouse-Condition

              13N-AS*

              13N-RAD†

              13N-CR‡

              15N-AS

              15N-RAD

              15N-CR

              13R-AS

              13R-RAD

              13R-CR

              Unfiltered Total Tags

              310,516

              318,102

              339,864

              338,210

              310,072

              326,870

              337,546

              314,440

              335,504

              No. of Bad Tags

              955

              1,010

              1,083

              1,097

              983

              737

              900

              744

              832

              Minus Bad Tags

                       

              Total Tags

              309,561

              317,092

              338,781

              337,113

              309,089

              326,133

              336,646

              313,696

              334,672

              Tag Types

              79,201

              96,973

              99,730

              81,850

              84,499

              88,249

              79,859

              91,438

              90,675

              No. of Duplicate Ditags

              19,761

              12,220

              12,678

              21,973

              17,471

              12,836

              24,552

              12,786

              13,127

              % of Duplicate Ditags

              6.38

              3.85

              3.74

              6.52

              5.65

              3.94

              7.29

              4.08

              3.92

              Average QF§ of Tags

              0.85

              0.88

              0.87

              0.86

              0.89

              0.88

              0.88

              0.80

              0.87

              No. of Tags QF < 0.95

              63,057

              62,872

              71,576

              68,993

              54,627

              54,470

              68,981

              101,215

              69,647

              Q ≥ 0.95

                       

              Total Tags

              246,504

              254,220

              267,205

              268,120

              254,462

              271,663

              267,665

              212,481

              265,025

              Tag Types

              52,033

              67,542

              66,748

              52,606

              59,374

              64,985

              53,715

              54,682

              64,837

              Total Tags Combined

                  

              2,307,345

                  

              Tag Types Combined

                  

              263,199

                  

              No. of LDTs II Type I

              124

              72

              174

              179

              84

              186

              164

              118

              301

              No. of LDTs Type II

              19

              9

              54

              56

              33

              40

              60

              24

              59

              Minus LDTs

                       

              Total Tags

              246,361

              254,139

              266,977

              267,885

              254,345

              271,437

              267,441

              212,339

              264,665

              Tag Types

              52,031

              67,540

              66,746

              52,604

              59,372

              64,983

              53,713

              54,680

              64,835

              Total Tags Combined

                  

              2,305,589

                  

              Tag Types Combined

                  

              263,197

                  

              * AS, Androgen-sensitive

              † RAD, Responsive to androgen-deprivation

              ‡ CR, Castration-recurrent

              § QF, Quality Factor

              II LDTs, Linker-derived tags

              The LongSAGE libraries were hierarchically clustered and displayed as a phylogenetic tree. In most cases, LongSAGE libraries made from the same disease stage (AS, RAD, or CR) clustered together more closely than LongSAGE libraries made from the same biological replicate (mice 13N, 15N, or 13R; Figure 1). This suggests the captured transcriptomes were representative of disease stage with minimal influence from biological variation.
              http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-3-43/MediaObjects/12920_2010_Article_178_Fig1_HTML.jpg
              Figure 1

              Clustering of the nine LongSAGE libraries in a hierarchical tree. The tree was generated using a Pearson correlation-based hierarchical clustering method and visualized with TreeView. LongSAGE libraries constructed from similar stages of prostate cancer progression (AS, androgen-sensitive; RAD, responsive to androgen-deprivation; and CR, castration-recurrent) cluster together. 13N, 15N, and 13R indicate the identity of each animal.

              Identification of groups of genes that behave similarly during progression of prostate cancer was conducted through K-means clustering of tags using the PoissonC algorithm [30]. For each biological replicate (mice 13N, 15N, or 13R), all tag types were clustered that had a combined count greater than ten in the three libraries representing disease stages (AS, RAD, and CR) and mapped unambiguously sense to a transcript in reference sequence (RefSeq; February 28th, 2008) [32] using DiscoverySpace4 software [33]. By plotting within cluster dispersion (i.e., intracluster variance) against a range of K (number of clusters; Additional file 1, Figure S2), we determined that ten clusters best embodied the expression patterns present in each biological replicate. This was decided based on the inflection point in the graph (Additional file 1, Figure S2), showing that after reaching K = 10, increasing the number of K did not substantially reduce the within cluster dispersion. K-means clustering was performed over 100 iterations, so that tags would be placed in clusters that best represent their expression trend. The most common clusters for each tag are displayed (Figure 2). In only three instances, there were similar clusters in just two of the three biological replicates. Consequently, consistent changes in gene expression during progression were represented in 11 patterns. Differences among expression patterns for each biological replicate may be explained by biological variation, the probability of sampling a given LongSAGE tag, and/or imperfections in K-means clustering (e.g, variance may not be a good measure of cluster scatter).
              http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-3-43/MediaObjects/12920_2010_Article_178_Fig2_HTML.jpg
              Figure 2

              K-means clustering of tag types with similar expression trends. PoissonC with K = 10 (where K = number of clusters) was conducted over 100 iterations separately for each biological replicate (mice 13N, 15N, and 13R) and the results from the iterations were combined into consensus clusters shown here. Plotted on the x-axes are the long serial analysis of gene expression (LongSAGE) libraries representing different stages of prostate progression: AS, androgen-sensitive; RAD, responsive to androgen-deprivation; and CR, castration-recurrent. Plotted on the y-axes are the relative expression levels of each tag type, represented as a percentage of the total tag count (for a particular tag type) in all three LongSAGE libraries. Different colors represent different tag types. Each of the ten clusters for each biological replicate are labeled as such. 'No equivalent' indicates that a similar expression trend was not observed in the indicated biological replicate. Eleven expression patterns are evident in total and are labeled on the left. K-means clusters were amalgamated into five major expression trends: group 1, up during progression; group 2, down during progression; group 3, peak in the RAD stage; group 4, constant during progression; and group 5, valley in RAD stage.

              Gene ontology enrichment analysis

              We conducted Gene Ontology (GO) [34] enrichment analysis using Expression Analysis Systematic Explorer (EASE) [35] software to determine whether specific GO annotations were over-represented in the K-means clusters. Enrichment was defined by the EASE score (p-value ≤ 0.05) generated during comparison to all the other clusters in the biological replicate. This analysis was done for each biological replicate (3 mice: 13N, 15N, or 13R).

              To enable visual differences between the 11 expression trends, the clusters were amalgamated into five major trends: group 1, up during progression; group 2, down during progression; group 3, peak in the RAD stage; group 4, constant during progression; and group 5, valley in RAD stage (Figure 2). To be consistent, the GO enrichment data was combined into five major trends which resulted in redundancy in GO terms. To simplify the GO enrichment data, similar terms were pooled into representative categories. Categorical gene ontology enrichments of the five major expression trends are shown in Figure 3. These data indicate that steroid binding, heat shock protein activity, de-phosphorylation activity, and glycolysis all decreased in the stage that was RAD, but increased again in the stage that was CR. Interestingly, steroid hormone receptor activity continues to increase throughout progression. Both of these expression trends were observed for genes with GO terms for transcription factor activity or secretion. The GO categories for genes with kinase activity and signal transduction displayed expression trends with peaks and valleys at the stage that was RAD. The levels of expression of genes involved in cell adhesion rose in the stage that was RAD, but dropped again in the stage that was CR.
              http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-3-43/MediaObjects/12920_2010_Article_178_Fig3_HTML.jpg
              Figure 3

              Gene Ontology enrichments of the five major expression trends. Plotted on the x-axis are Gene Ontology (GO) categories enriched in one or more of the five major expression trends. On the z-axis the five major expression trends correspond to Figure 2 and are: group 1, up during progression; group 2, down during progression; group 3, peak in the RAD stage; group 4, constant during progression; and group 5, valley in RAD stage. The y-axis displays the number of biological replicates (number of mice: 1, 2, or 3) exhibiting enrichment. The latter allows one to gauge the magnitude of the GO enrichment and confidence.

              Altogether, genes with functional categories that were enriched in expression trends may be consistent with the AR signaling pathway playing a role in progression of prostate cancer to castration-recurrence (Figure 3). For example, GO terms steroid binding, steroid hormone receptor activity, heat shock protein activity, chaperone activity, and kinase activity could represent the cytoplasmic events of AR signaling. GO terms transcription factor activity, regulation of transcription, transcription corepression activity, and transcription co-activator activity could represent the nuclear events of AR signaling. AR-mediated gene transcription may result in splicing and protein translation, to regulate general cellular processes such as proliferation (and related nucleotide synthesis, DNA replication, oxidative phosphorylation, oxioreductase activity, and glycolysis), secretion, and differentiation.

              It should be noted, however, that both positive and negative regulators were represented in the GO enriched categories (Figure 3). Therefore, a more detailed analysis was required to determine if the pathways represented by the GO-enriched categories were promoted or inhibited during progression to CRPC. Moreover, many of the GO enrichments that were consistent with changes in the AR signaling pathway were generic, and could be applied to the other models of CRPC.

              Consistent differential gene expression associated with progression of prostate cancer

              Pair-wise comparisons were made between LongSAGE libraries representing the transcriptomes of different stages (AS, RAD, and CR) of prostate cancer progression from the same biological replicate (3 mice: 13N, 15N, or 13R). Among all three biological replicates, the number of consistent statistically significant differentially expressed tag types were determined using the Audic and Claverie test statistic [36] at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001 (Table 2). The tags represented in Table 2 were included only if the associated expression trend was common among all three biological replicates. The Audic and Claverie statistical method is well-suited for LongSAGE data, because the method takes into account the sizes of the libraries and tag counts. Tag types were counted multiple times if they were over, or under-represented in more than one comparison. The number of tag types differentially expressed decreased by 57% as the stringency of the p-value increased from p ≤ 0.05 to 0.001.
              Table 2

              Number of tag types consistently and significantly differentially expressed among all three biological replicates and between conditions*

              Comparison

              Change

              p ≤ 0.001

              p ≤ 0.01

              p ≤ 0.05

              AS† vs. RAD‡

              Up in RAD

              21

              44

              83

               

              Down in RAD

              68

              105

              149

               

              Total

              89

              149

              232

              RAD vs. CR§

              Up in CR

              24

              45

              89

               

              Down in CR

              46

              59

              104

               

              Total

              70

              104

              193

              AS vs. CR

              Up in CR

              111

              167

              294

               

              Down in CR

              127

              168

              256

               

              Total

              238

              335

              550

              * Statistics according to the Audic and Claverie test statistic

              † AS, Androgen-sensitive

              ‡ RAD, Responsive to androgen-deprivation

              § CR, Castration-recurrent

              Tag types consistently differentially expressed in pair-wise comparisons were mapped to RefSeq (March 4th, 2008). Tags that mapped anti-sense to genes, or mapped ambiguously to more than one gene were not included in the functional analysis. GO, Kyoto Encyclopedia of Genes and Genomes (KEGG; v45.0) [37] pathway, and SwissProt (v13.0) [38] keyword annotation enrichment analyses were conducted using EASE (v1.21; March 11th, 2008) and FatiGO (v3; March 11th, 2008) [39] (Table 3). This functional analysis revealed that the expression of genes involved in signaling increased during progression, but the expression of genes involved in protein synthesis decreased during progression. Cell communication increased in the stage that was RAD but leveled off in the stage that was CR. Carbohydrate, lipid and amino acid synthesis was steady in the RAD stage but increased in the CR stage. Lastly, glycolysis decreased in the RAD stage, but was re-expressed in the CR stage (Table 3).
              Table 3

              Top five enrichments of functional categories of tags consistently and significantly differentially expressed among all three biological replicates and between stages of prostate cancer*

              Top 5 GO † categories

              P-value ‡

              Top 5 KEGG § annotations

              P-value II

              Top 5 SwissProt annotations

              P-value II

              AS vs. RAD: Up in RAD¶

              Cell communication

              2.E-02

              Stilbene, coumarine and lignin biosynthesis

              1.E-02

              Antioxidant

              7.E-04

              Extracellular

              2.E-02

              Butanoate metabolism

              2.E-02

              Cell adhesion

              5.E-03

              Extracellular matrix

              2.E-02

              2,4-Dichlorobenzoate degradation

              2.E-02

              Signal

              6.E-03

              Synaptic vesicle transport

              3.E-02

              Cell adhesion molecules (CAMs)

              2.E-02

              Fertilization

              7.E-03

              Synapse

              4.E-02

              Alkaloid biosynthesis II

              5.E-02

              Amyotrophic lateral sclerosis

              7.E-03

              AS vs. RAD: Down in RAD

              Glycolysis

              3.E-05

              Glycolysis/Gluconeogenesis

              3.E-05

              Glycolysis

              3.E-07

              Glucose catabolism

              1.E-04

              Ribosome

              2.E-03

              Pyrrolidone carboxylic acid

              8.E-05

              Hexose catabolism

              1.E-04

              Carbon fixation

              3.E-03

              Pyridoxal phosphate

              2.E-04

              Hexose metabolism

              2.E-04

              Fructose and mannose metabolism

              2.E-02

              Gluconeogenesis

              3.E-04

              Monosaccharide catabolism

              2.E-04

              Urea cycle and metabolism of amino groups

              3.E-02

              Coiled coil

              5.E-03

              RAD vs. CR: Up in CR

              Acid phosphatase activity

              4.E-02

              gamma-Hexachlorocyclohexane degradation

              5.E-03

              Lyase

              2.E-03

              Lyase activity**

              7.E-02

              Glycolysis/Gluconeogenesis

              3.E-02

              Immune response

              5.E-03

              Carbohydrate metabolism**

              9.E-02

              O-Glycan biosynthesis

              5.E-02

              Signal

              6.E-03

              Extracellular**

              1.E-01

              Ether lipid metabolism**

              6.E-02

              Glycolysis

              7.E-03

              Catabolism**

              1.E-01

              Phenylalanine, tyrosine and tryptophan biosynthesis**

              6.E-02

              Progressive external ophthalmoplegia

              1.E-02

              RAD vs. CR: Down in CR

              Cytosolic ribosome

              2.E-09

              Ribosome

              2.E-11

              Ribosomal protein

              6.E-10

              Large ribosomal subunit

              1.E-07

              Urea cycle and metabolism of amino groups

              1.E-02

              Ribonucleoprotein

              3.E-08

              Cytosol

              2.E-07

              Arginine and proline metabolism

              4.E-02

              Acetylation

              1.E-05

              Cytosolic large ribosomal subunit

              2.E-07

              Type II diabetes mellitus**

              1.E-01

              Elongation factor

              1.E-03

              Protein biosynthesis

              2.E-07

              Phenylalanine metabolism**

              1.E-01

              rRNA-binding

              2.E-03

              AS vs. CR: Up in CR

              Synapse

              4.E-03

              Butanoate metabolism

              2.E-03

              Glycoprotein

              2.E-03

              Extracellular

              5.E-03

              Ascorbate and aldarate metabolism

              2.E-02

              Vitamin C

              7.E-03

              Transition metal ion binding

              7.E-03

              Phenylalanine metabolism

              2.E-02

              Lipoprotein

              1.E-02

              Metal ion binding

              2.E-02

              Linoleic acid metabolism

              2.E-02

              Signal

              1.E-02

              Extracellular matrix

              2.E-02

              gamma-Hexachlorocyclohexane degradation

              2.E-02

              Heparin-binding

              1.E-02

              AS vs. CR: Down in CR

              Cytosolic ribosome

              4.E-12

              Ribosome

              2.E-09

              Acetylation

              2.E-07

              Biosynthesis

              7.E-11

              Carbon fixation

              9.E-04

              Ribosomal protein

              1.E-06

              Macromolecule biosynthesis

              2.E-10

              Glycolysis/Gluconeogenesis

              3.E-03

              Glycolysis

              7.E-05

              Protein biosynthesis

              1.E-08

              Glycosphingolipid biosynthesis - lactoseries

              4.E-02

              Ribonucleoprotein

              8.E-05

              Eukaryotic 43 S preinitiation complex

              2.E-08

              Glutamate metabolism**

              8.E-02

              Protein biosynthesis

              1.E-04

              * Statistics according to the Audic and Claverie test statistic (p ≤ 0.05)

              † GO, Gene Ontology

              ‡ P-value represents the raw EASE (Expression Analysis Systematic Explorer) score

              § KEGG, Kyoto Encyclopedia of Genes and Genomes

              II Unadjusted p-value was computed using FatiGO

              ¶ AS, androgen-sensitive; RAD, responsive to androgen-deprivation; CR, castration-recurrent

              ** Not statistically significant (p > 0.05)

              Tag types differentially expressed between the RAD and CR stages of prostate cancer were of particular interest (Table 4). This is because these tags potentially represent markers for CRPC and/or are involved in the mechanisms of progression to CRPC. These 193 tag types (Table 2) were mapped to databases RefSeq (July 9th, 2007), Mammalian Gene Collection (MGC; July 9th, 2007) [40], or Ensembl Transcript or genome (v45.36d) [41]. Only 135 of the 193 tag types were relevant (Table 4) with 48 tag types that mapped ambiguously to more than one location in the Homo Sapiens transcriptome/genome, and another 10 tag types that mapped to Mus musculus transcriptome/genome. Mus musculus mappings may be an indication of minor contamination of the in vivo LNCaP Hollow Fiber model samples with host (mouse) RNA. These 135 tag types represented 114 candidate genes with 7 tag types that did not map to the genome, 5 tag types that mapped to unannotated genomic locations, and 9 genes that were associated with more than one tag type. Table 4 shows the LongSAGE tag sequences and tag counts per million tags in all nine libraries. Tags were sorted into groups based on expression trends. These trends are visually represented in Additional file 1, Figure S3. Mapping information was provided where available.
              Table 4

              Gene expression trends of LongSAGE tags that consistently and significantly altered expression in CR prostate cancer*

               

              13N

              15N

              13R

                 
               

              AS§

              RADII

              CR¶

              AS

              RAD

              CR

              AS

              RAD

              CR

                 

              Tag Sequence

              S1885

              S1886

              S1887

              S1888

              S1889

              S1890

              S1891

              S1892

              S1893

              Trend‡

              Gene**

              Accession§§

              TCTAGAGAACACTGTGC

              12†

              79

              382

              7

              67

              136

              7

              52

              200

              A

              ACPP‡‡

              NM_001099

              TAATTTTTCTAAGGTGT

              101

              311

              648

              119

              397

              895

              120

              546

              918

              A

              C1ORF80

              ENSG00000186063

              TGAGAGAGGCCAGAACA

              8

              39

              150

              4

              39

              144

              7

              33

              95

              A

              N/A

              Genomic

              CTCATAAGGAAAGGTTA

              637

              952

              1680

              653

              1170

              1540

              688

              1620

              1930

              A

              RNF208

              BC090061

              GATTTCTATTTGTTTTT

              89

              169

              446

              116

              208

              339

              86

              311

              555

              A

              SERINC5

              ENSG00000164300

              GTTGGGAAGACGTCACC

              426

              571

              742

              273

              417

              741

              262

              363

              495

              A

              STEAP1

              NM_012449

              GAGGATCACTTGAGGCC

              191

              299

              449

              134

              189

              589

              187

              203

              314

              B

              AMACR‡‡

              BC009471

              TTGTTGATTGAAAATTT

              219

              197

              528

              273

              197

              479

              232

              391

              586

              B

              AMD1‡‡

              NM_001634

              TTTGCTTTTGTTTTGTT

              53

              16

              169

              34

              51

              129

              7

              28

              72

              B

              AQP3

              NM_004925

              GTTCGACTGCCCACCAG

              45

              28

              101

              52

              47

              122

              34

              42

              106

              B

              ASAH1††

              NM_177924

              TAATAAACAGGTTTTTA

              426

              232

              648

              332

              315

              700

              138

              250

              491

              B

              ASAH1‡‡

              NM_177924

              TCACAGCTGTGAAGATC

              85

              110

              277

              161

              71

              258

              310

              438

              945

              B

              BTG1

              NM_001731

              AAAAGAGAAAGCACTTT

              24

              75

              199

              19

              35

              85

              15

              90

              552

              B

              CAMK2N1

              NM_018584

              CAAAACAGGCAGCTGGT

              4

              71

              169

              15

              83

              162

              37

              75

              268

              B

              CAMK2N1††

              NM_018584

              AGGAGGAAGAATGGACT

              33

              59

              187

              49

              67

              247

              26

              42

              223

              B

              CCNH

              NM_001239

              TTTTAAAAATATAAAAT

              89

              83

              243

              97

              130

              269

              64

              170

              382

              B

              COMT

              NM_000754

              GAATGAAATAAAAAATA

              134

              252

              626

              209

              240

              357

              116

              160

              272

              B

              DHRS7

              NM_016029

              AAAGTGCATCCTTTCCC

              118

              146

              318

              153

              220

              394

              288

              231

              646

              B

              FGFRL1

              NM_001004356

              AAACTGAATAAGGAGAA

              24

              51

              236

              19

              51

              438

              19

              146

              283

              B

              GALNT3

              NM_004482

              TTTAAGGAAACATTTGA

              4

              4

              75

              4

              4

              81

              0

              0

              57

              B

              GALNT3††

              NM_004482

              CCAACCGTGCTTGTACT

              191

              327

              521

              202

              279

              534

              172

              363

              510

              B

              GLO1

              NM_006708

              GAGGGCCGGTGACATCT

              300

              378

              1170

              321

              476

              1230

              254

              447

              1030

              B

              H2AFJ

              NM_177925

              TATCATTATTTTTACAA

              57

              63

              161

              67

              63

              181

              75

              94

              181

              B

              HSD17B4

              NM_000414

              AATGCACTTATGTTTGC

              16

              8

              64

              22

              16

              77

              19

              28

              98

              B

              N/A

              No map

              ACCTTCGCAGGGGAGAG

              0

              0

              19

              0

              4

              41

              0

              5

              34

              B

              N/A

              Genomic

              ATAACCTGAAAGGAAAG

              0

              16

              56

              7

              4

              74

              0

              28

              87

              B

              N/A

              No map

              GTGATGTGCACCTGTTG

              0

              0

              38

              4

              0

              30

              0

              5

              45

              B

              N/A

              No map

              GTTTGGAGGTACTAAAG

              20

              43

              94

              34

              87

              169

              34

              90

              234

              B

              N/A

              Genomic

              TTTTCAAAAATTGGAAA

              0

              35

              180

              7

              4

              59

              0

              19

              61

              B

              N/A

              No map

              GAAAAATTTAAAGCTAA

              394

              397

              569

              433

              598

              788

              853

              862

              1060

              B

              NGFRAP1

              NM_206917

              CAAATTCAGGGAGCACA

              0

              4

              139

              4

              16

              228

              0

              14

              136

              B

              OPRK1

              NM_000912

              CTATTGTCTGAACTTGA

              0

              8

              109

              0

              12

              70

              0

              9

              227

              B

              OR51E2

              BC020768

              ATGCTAATTATGGCAAT

              4

              12

              75

              4

              8

              74

              0

              5

              57

              B

              PCGEM1

              NR_002769

              CAGAAAGCATCCCTCAC

              4

              43

              195

              0

              16

              111

              7

              33

              264

              B

              PLA2G2A‡‡

              NM_000300

              TAATTTTAGTGCTTTGA

              16

              75

              154

              37

              59

              162

              4

              57

              132

              B

              PTGFR

              NM_000959

              TTGTTTGTAAATAGAAT

              0

              12

              94

              0

              4

              162

              0

              14

              72

              B

              QKI

              NM_206853

              TAAACACTGTAAAATCC

              0

              4

              75

              0

              4

              66

              0

              0

              42

              B

              QKI††

              NM_206853

              AGCAGATCAGGACACTT

              20

              35

              112

              15

              16

              140

              15

              42

              98

              B

              S100A10

              NM_002966

              CTGCCATAACTTAGATT

              37

              55

              161

              93

              63

              192

              56

              99

              264

              B

              SBDS

              NM_016038

              TGGCTGAGTTTATTTTT

              20

              24

              79

              41

              8

              96

              4

              42

              147

              B

              SFRS2B

              NM_032102

              GAAGATTAATGAGGGAA

              126

              142

              277

              108

              130

              402

              101

              188

              325

              B

              SNX3

              NM_003795

              ATGGTACTAAATGTTTT

              16

              47

              124

              37

              28

              88

              11

              19

              76

              B

              SPIRE1

              NM_020148

              TATATATTAAGTAGCCG

              45

              39

              101

              45

              75

              133

              41

              75

              178

              B

              STEAP2‡‡

              NM_152999

              CAACAATATATGCTTTA

              24

              32

              82

              75

              32

              136

              26

              99

              212

              B

              STEAP2††

              NM_152999

              TTTCATTGCCTGAATAA

              24

              43

              150

              34

              59

              114

              22

              61

              178

              B

              TACC1‡‡

              NM_006283

              TTGGCCAGTCTGCTTTC

              8

              16

              67

              4

              4

              77

              0

              5

              38

              B

              TMEM30A

              ENSG00000112697

              ATATCACTTCTTCTAGA

              12

              4

              26

              7

              4

              26

              0

              52

              140

              C

              ADAM2‡‡

              NM_001464

              ATGTGTGTTGTATTTTA

              812

              338

              768

              1010

              315

              1020

              269

              702

              865

              C

              BNIP3

              NM_004052

              CCACGTTCCACAGTTGC

              601

              291

              599

              530

              346

              700

              381

              339

              559

              C

              ENO2

              NM_001975

              CTGATCTGTGTTTCCTC

              16

              0

              26

              0

              4

              41

              19

              0

              34

              C

              HLA-B

              BC013187

              AGCCCTACAAACAACTA

              382

              441

              596

              508

              456

              619

              400

              631

              1010

              C

              MT-ND3

              ENSG00000198840

              ATATTTTCTTTGTGGAA

              20

              12

              90

              7

              0

              48

              4

              0

              23

              C

              N/A

              No map

              CAAGCATCCCCGTTCCA

              2400

              2130

              2440

              2730

              1720

              2250

              1020

              2010

              2340

              C

              N/A

              ENSG00000211459

              GTTGTAAAATAAACTTT

              118

              83

              172

              228

              87

              247

              112

              203

              378

              C

              N/A

              Genoic

              TTGGATTTCCAAAGCAG

              12

              0

              19

              0

              0

              33

              0

              0

              26

              C

              N/A

              Genomic

              TCTTTTAGCCAATTCAG

              138

              181

              420

              381

              326

              468

              389

              334

              457

              C

              NKX3-1††

              NM_006167

              TGATTGCCCTTTCATAT

              73

              39

              86

              86

              39

              107

              108

              99

              181

              C

              P4HA1

              NM_000917

              GTAACAAGCTCTGGTAT

              28

              16

              56

              49

              24

              66

              11

              19

              72

              C

              PJA2

              NM_014819

              ACAGTGCTTGCATCCTA

              85

              75

              139

              108

              98

              203

              101

              118

              196

              C

              PPP2CB

              NM_004156

              AGGCGAGATCAATCCCT

              57

              39

              101

              37

              24

              122

              131

              66

              268

              C

              PSMA7

              NM_002792

              TATTTTGTATTTATTTT

              73

              59

              180

              93

              51

              111

              22

              94

              253

              C

              SLC25A4

              NM_001151

              TTATGGATCTCTCTGCG

              1050

              1260

              1820

              1140

              1300

              2260

              1990

              1010

              1530

              C

              SPON2

              NM_012445

              CAGTTCTCTGTGAAATC

              767

              515

              1060

              855

              503

              914

              467

              608

              1200

              C

              TMEM66

              NM_016127

              AAATAAATAATGGAGGA

              138

              59

              255

              82

              118

              284

              165

              90

              159

              C

              TRPM8

              NM_024080

              ATGTTTAATTTTGCACA

              61

              87

              154

              157

              59

              195

              217

              85

              344

              C

              WDR45L

              NM_019613

              GGGCCCCAAAGCACTGC

              861

              543

              1180

              1020

              657

              1590

              1240

              739

              937

              E

              C19orf48

              NM_199249

              TCCCCGTGGCTGTGGGG

              1670

              1390

              2290

              1740

              1410

              1720

              3370

              970

              1180

              E

              DHCR24‡‡

              BC004375

              GCATCTGTTTACATTTA

              487

              201

              345

              444

              208

              468

              684

              226

              423

              E

              ELOVL5

              NM_021814

              GAAATTAGGGAAGCCTT

              317

              153

              311

              310

              181

              542

              359

              193

              298

              E

              ENDOD1

              XM_290546

              GGATGGGGATGAAGTAA

              2780

              1160

              4780

              2950

              1350

              3620

              2930

              1230

              1890

              E

              KLK3‡‡

              NM_001648

              TGAAAAGCTTAATAAAT

              313

              142

              322

              474

              181

              332

              273

              179

              314

              E

              TPD52

              NM_001025252

              GTTGTGGTTAATCTGGT

              1770

              634

              1270

              1800

              806

              1190

              2480

              659

              960

              F

              B2M

              NM_004048

              GAAACAAGATGAAATTC

              4380

              1170

              2260

              5300

              1110

              2720

              3750

              2220

              2830

              F

              PGK1

              NM_000291

              AGCACCTCCAGCTGTAC

              2150

              1130

              648

              2060

              1560

              939

              1560

              1200

              722

              G

              EEF2

              NM_001961

              GCACAAGAAGATTAAAA

              536

              228

              124

              762

              425

              195

              838

              278

              174

              G

              GAS5

              NR_002578

              CCGCTGCGTGAGGGCAG

              451

              169

              56

              429

              197

              44

              516

              94

              0

              G

              HES6

              NM_018645

              GCCCAGGTCACCCACCC

              585

              55

              4

              519

              79

              7

              456

              66

              0

              G

              LOC644844

              XM_927939

              ATGCAGCCATATGGAAG

              2650

              386

              82

              2470

              216

              129

              1210

              259

              98

              G

              ODC1

              NM_002539

              CGCTGGTTCCAGCAGAA

              1420

              811

              479

              1250

              959

              553

              800

              589

              374

              G

              RPL11

              NM_000975

              AAGACAGTGGCTGGCGG

              2650

              1730

              1220

              2460

              1860

              1350

              2120

              1630

              1270

              G

              RPL37A‡‡

              NM_000998

              TTCTTGTGGCGCTTCTC

              925

              543

              217

              1030

              708

              273

              1130

              419

              306

              G

              RPS11††

              NM_001015

              GGTGAGACACTCCAGTA

              463

              252

              165

              485

              346

              192

              363

              245

              159

              G

              SLC25A6

              NM_001636

              AGGTTTTGCCTCATTCC

              982

              515

              281

              1200

              491

              243

              688

              782

              166

              H

              ABHD2

              NM_007011

              TGAAGGAGCCGTCTCCA

              317

              272

              187

              392

              295

              199

              366

              259

              140

              H

              ATP5G2

              NM_001002031

              CTCAGCAGATCCAAGAG

              191

              185

              67

              254

              232

              66

              142

              231

              79

              H

              C17orf45

              NM_152350

              CTGTGACACAGCTTGCC

              308

              397

              172

              209

              307

              125

              295

              226

              110

              H

              CCT2

              NM_006431

              TCTGCACCTCCGCTTGC

              495

              606

              277

              426

              570

              276

              366

              471

              204

              H

              EEF1A2

              NM_001958

              GCCCAAGGACCCCCTGC

              114

              114

              38

              138

              98

              41

              101

              42

              4

              H

              FLNA‡‡

              NM_001456

              TTATGGGATCTCAACGA

              564

              425

              180

              642

              452

              317

              430

              490

              253

              H

              GNB2L1

              NM_006098

              TCTGCAAAGGAGAAGTC

              81

              102

              38

              105

              87

              26

              165

              80

              30

              H

              HMGB2

              NM_002129

              CTTGTGAACTGCACAAC

              268

              228

              124

              231

              177

              103

              273

              160

              57

              H

              HN1

              NM_016185

              TCTGAAGTTTGCCCCAG

              313

              291

              150

              254

              299

              155

              187

              226

              72

              H

              MAOA

              NM_000240

              TTAATTGATAGAATAAA

              483

              350

              199

              422

              287

              103

              273

              235

              83

              H

              MAOA

              NM_000240

              GGCAGCCAGAGCTCCAA

              1200

              1260

              420

              1050

              672

              350

              681

              819

              23

              H

              MARCKSL1

              NM_023009

              CCCTGCCTTGTCCCTCT

              353

              240

              112

              310

              263

              107

              176

              193

              102

              H

              MDK

              NM_001012334

              CTGTGGATGTGTCCCCC

              649

              476

              169

              459

              389

              214

              430

              297

              117

              H

              N/A

              No map

              CTCCTCACCTGTATTTT

              1120

              771

              262

              1220

              979

              313

              666

              730

              261

              H

              RPL13A‡‡

              NM_012423

              GCAGCCATCCGCAGGGC

              1980

              1770

              809

              2300

              1730

              928

              2150

              1570

              1020

              H

              RPL28

              NM_000991

              GGATTTGGCCTTTTTGA

              3470

              2070

              1370

              4170

              2910

              1540

              2800

              2870

              2500

              H

              RPLP2‡‡

              NM_001004

              TCTGTACACCTGTCCCC

              2320

              1670

              850

              1930

              1880

              825

              2130

              1490

              1120

              H

              RPS11

              NM_001015

              GCTTTTAAGGATACCGG

              1510

              1050

              626

              1860

              1120

              593

              1550

              1550

              960

              H

              RPS20‡‡

              NM_001023

              CCCCAGCCAGTCCCCAC

              921

              519

              281

              788

              664

              357

              1100

              438

              291

              H

              RPS3

              NM_001005

              CCCCCAATGCTGAGGCC

              89

              138

              26

              90

              94

              30

              90

              80

              30

              H

              SF3A2

              NM_007165

              GCCGCCATCTCCGAGAG

              195

              102

              30

              168

              118

              55

              172

              108

              30

              H

              TKT

              NM_001064

              GGCCATCTCTTCCTCAG

              349

              307

              202

              317

              346

              173

              277

              254

              121

              H

              YWHAQ

              NM_006826

              AGGCTGTGTTCCTCCGT

              16

              39

              11

              34

              67

              22

              26

              38

              8

              I

              ACY1

              NM_000666

              TGCCTCTGCGGGGCAGG

              446

              649

              427

              399

              664

              424

              501

              462

              317

              I

              CD151

              NM_004357

              GGCACAGTAAAGGTGGC

              175

              216

              142

              332

              350

              173

              456

              316

              204

              I

              CUEDC2

              NM_024040

              TCACACAGTGCCTGTCG

              49

              71

              7

              30

              47

              15

              34

              66

              4

              I

              CXCR7

              NM_001047841

              TGTGAGGGAAGCTGCTT

              53

              87

              15

              67

              102

              52

              52

              90

              42

              I

              FKBP10

              BC016467

              TGCTTTGCTTCATTCTG

              28

              63

              26

              22

              79

              26

              49

              118

              61

              I

              GRB10

              NM_005311

              GTACTGTATGCTTGCCA

              170

              212

              82

              134

              153

              88

              123

              188

              113

              I

              KPNB1‡‡

              NM_002265

              GTGGCAGTGGCCAGTTG

              106

              193

              97

              123

              173

              96

              94

              137

              76

              I

              N/A

              ENSG00000138744

              GGGGAGCCCCGGGCCCG

              61

              63

              26

              30

              51

              18

              34

              57

              0

              I

              NAT14

              NM_020378

              TGTTCAGGACCCTCCCT

              28

              67

              26

              60

              63

              26

              60

              28

              0

              I

              NELF

              NM_015537

              TTTTCCTGGGGATCCTC

              41

              130

              15

              37

              87

              33

              56

              104

              45

              I

              PCOTH

              NM_001014442

              GAAACCCGGTAGTCTAG

              41

              75

              4

              37

              75

              26

              52

              151

              30

              I

              PLCB4

              NM_000933

              GTCTGACCCCAGGCCCC

              126

              205

              82

              119

              193

              103

              157

              179

              38

              I

              PPP2R1A

              NM_014225

              GGCCCGAGTTACTTTTC

              231

              150

              75

              161

              232

              136

              142

              160

              45

              I

              RPL35A††

              NM_000996

              GTTCGTGCCAAATTCCG

              881

              696

              390

              1100

              712

              523

              497

              782

              461

              I

              RPL35A‡‡

              NM_000996

              TTACCATATCAAGCTGA

              877

              535

              311

              1130

              598

              405

              636

              791

              578

              I

              RPL39‡‡

              NM_001000

              GCTGCAGCACAAGCGGC

              268

              244

              127

              45

              216

              125

              157

              71

              11

              I

              RPS18††

              NM_022551

              AGCTCTTGGAGGCACCA

              203

              319

              206

              142

              421

              243

              269

              259

              162

              I

              SELENBP1

              NM_003944

              TGCTGGTGTGTAAGGGG

              69

              102

              45

              82

              87

              37

              105

              75

              30

              I

              SH3BP5L

              NM_030645

              GAGAGTAACAGGCCTGC

              191

              150

              71

              112

              181

              111

              108

              165

              64

              I

              SYNC1

              NM_030786

              CTGAAAACCACTCAAAC

              394

              508

              225

              306

              547

              236

              310

              381

              200

              I

              TFPI

              NM_006287

              TAAAAAAGGTTTCATCC

              183

              248

              127

              86

              130

              66

              142

              268

              87

              I

              TFPI

              NM_006287

              CTCCCTCCTCTCCTACC

              28

              32

              4

              30

              39

              7

              71

              24

              0

              I

              TK1

              NM_003258

              CATTTTCTAATTTTGTG

              544

              744

              236

              407

              771

              181

              288

              664

              185

              J

              N/A

              No map

              TGATTTCACTTCCACTC

              3480

              5260

              3910

              3700

              6110

              3590

              3040

              5960

              2600

              K

              MT-CO3

              ENSG00000198938

              TTTCTGTCTGGGGAAGG

              130

              236

              82

              123

              201

              111

              101

              188

              113

              K

              PIK3CD

              NM_005026

              GCCGCTACTTCAGGAGC

              256

              370

              199

              224

              330

              169

              142

              316

              38

              K

              RAMP1

              NM_005855

              ATGGTTACACTTTTGGT

              93

              161

              94

              75

              208

              118

              60

              226

              95

              K

              UTX

              NM_021140

              CACTACTCACCAGACGC

              2820

              3900

              3020

              2740

              4290

              2440

              2620

              3120

              1260

              K

              VPS13B††

              ENSG00000132549

              CTAAGACTTCACCAGTC

              7120

              11000

              9730

              6390

              10900

              8330

              3610

              8870

              7850

              L

              N/A

              ENSG00000210082

              * Statistics according to the Audic and Claverie test statistic (p ≤ 0.05)

              † Tag count per 1 million = (observed tag count/total tags in the library) × 1,000,000

              ‡ Trends are visually represented from A to P in Additional file 1, Figure S3. In addition to p-value considerations, significantly different trends were also required to display uniform directions of change in each biological replicate.

              § AS, Androgen-sensitive

              II RAD, Responsive to androgen-deprivation

              ¶ CR, Castration-recurrent

              ** Human Genome Nomenclature Committee (HGNC)-approved gene names were used when possible. Non-HGNC-approved gene names were not italicized.

              †† Tag maps antisense to gene

              ‡‡ Gene is known to display this expression trend in castration-recurrence

              §§ Accession numbers were displayed following the priority (where available): RefSeq > Mammalian Gene Collection > Ensembl Gene. If the tag mapped to more than one transcript variant of the same gene, the accession number of the lowest numerical transcript variant was displayed.

              We cross-referenced these 114 candidate genes with 28 papers that report global gene expression analyses on tissue samples from men with 'castration-recurrent', 'androgen independent,' 'hormone refractory,' 'androgen-ablation resistant,' 'relapsed,' or 'recurrent' prostate cancer, or animal models of castration-recurrence [4269]. The candidate genes were identified with HUGO Gene Nomenclature Committee (HGNC) approved gene names, aliases, descriptions, and accession numbers. The gene expression trends of 18 genes of 114 genes were previously associated with CRPC. These genes were: ACPP, ADAM2, AMACR, AMD1, ASAH1, DHCR24, FLNA, KLK3, KPNB1, PLA2G2A, RPL13A, RPL35A, RPL37A, RPL39, RPLP2, RPS20, STEAP2, and TACC (Table 4). To our knowledge, the gene expression trends of the remaining 96 genes have never before been associated with CRPC (Tables 4 &5).

              A literature search helped to gauge the potential of these 96 genes to be novel biomarkers or therapeutic targets of CRPC. The results of this literature search are presented in Table 5. We found 31 genes that encode for protein products that are known, or predicted, to be plasma membrane bound or secreted extracellularly (Bioinformatic Harvester). These genes were: ABHD2, AQP3, B2 M, C19orf48, CD151, CXCR7, DHRS7, ELOVL5, ENDOD1, ENO2, FGFRL1, GNB2L1, GRB10, HLA-B, MARCKSL1, MDK, NAT14, NELF, OPRK1, OR51E2, PLCB4, PTGFR, RAMP1, S100A10, SPON2, STEAP1, TFPI, TMEM30A, TMEM66, TRPM8, and VPS13B. Secretion of a protein could facilitate detection of the putative biomarkers in blood, urine, or biopsy sample. Twenty-one of the candidate genes are known to alter their levels of expression in response to androgen. These genes were: ABHD2, B2 M, BTG1, C19orf48, CAMK2N1, CXCR7, EEF1A2, ELOVL5, ENDOD1, HSD17B4, MAOA, MDK, NKX3-1, ODC1, P4HA1, PCGEM1, PGK1, SELENBP1, TMEM66, TPD52, and TRPM8 [9, 22, 7081]. Genes regulated by androgen may be helpful in determining the activation status of AR in CRPC. Enriched expression of a protein in prostate tissue could be indicative of whether a tumor is of prostatic origin. Eight of these 96 genes are known to be over-represented in prostate tissue [75, 8285]. These genes were: ELOVL5, NKX3-1, PCGEM1, PCOTH, RAMP1, SPON2, STEAP1, and TPD52. Twenty-six genes (ABHD2, BNIP3, EEF1A2, ELOVL5, GALNT3, GLO1, HSD17B4, MARCKSL1, MDK, NGFRAP1, ODC1, OR51E2, PCGEM1, PCOTH, PGK1, PP2CB, PSMA7, RAMP1, RPS18, SELENBP1, SLC25A4, SLC25A6, SPON2, STEAP1, TPD52, and TRPM8) have known associations to prostate cancer [57, 82, 86102]. Six genes (C1orf80, CAMK2N1, GLO1, MAOA, PGK1, and SNX3) have been linked to high Gleason grade [58, 103, 104], and twelve genes (B2 M, CAMK2N1, CD151, COMT, GALNT3, GLO1, ODC1, PCGEM1, PCOTH, SBDS, TMEM30A, and TPD52) have been implicated in the 'progression' of prostate cancer [58, 82], and 15 more genes (CD151, CXCR7, DHRS7, GNB2L1, HES6, HN1, NKX3-1, PGK1, PIK3CD, RPL11, RPS11, SF3A2, TK1, TPD52, and VPS13B) in the metastasis of prostate cancer [105, 106].
              Table 5

              Characteristics of genes with novel association to castration-recurrence in vivo

                  

              Associated with

                  

              Associated with

              Gene*

              S or PM†

              Reg. by A‡

              Spec. to P§

              CaPII

              GG¶

              Prog.**

              Mets††

              CR‡‡

              Gene

              S or PM

              Reg. by A

              Spec. to P

              CaP

              GG

              Prog.

              Mets

              CR

              ABHD2

              PM

              Y↑

              -

              Y↑

              -

              -

              -

              -

              NKX3-1

              -

              Y↑

              Y

              -

              -

              -

              Y

              -

              ACY1

              -

              -

              -

              -

              -

              -

              -

              -

              ODC1

              -

              Y↑

              -

              Y↑

              -

              Y↓

              -

              Y↑

              AQP3

              PM

              -

              -

              -

              -

              -

              -

              -

              OPRK1

              PM

              -

              -

              -

              -

              -

              -

              -

              ATP5G2

              -

              -

              -

              -

              -

              -

              -

              -

              OR51E2

              PM

              -

              -

              Y↑

              -

              -

              -

              -

              B2M

              S&PM

              Y↑

              -

              -

              -

              Y↑

              -

              Y↓

              P4HA1

              -

              Y

              -

              -

              -

              -

              -

              -

              BNIP3

              -

              -

              -

              Y↓

              -

              -

              -

              -

              PCGEM1

              -

              Y↑

              Y

              Y↑

              -

              Y↑

              -

              -

              BTG1

              -

              Y↓

              -

              -

              -

              -

              -

              -

              PCOTH

              -

              -

              Y

              Y↑

              -

              Y↑

              -

              -

              C17orf45

              -

              -

              -

              -

              -

              -

              -

              -

              PGK1

              -

              Y↑

              -

              Y↓

              Y↑

              -

              Y ↑↓§§

              -

              C19orf48

              S

              Y↑

              -

              -

              -

              -

              -

              -

              PIK3CD

              -

              -

              -

              -

              -

              -

              Y↑

              Y↑

              C1orf80

              -

              -

              -

              -

              Y↑

              -

              -

              -

              PJA2

              -

              -

              -

              -

              -

              -

              -

              -

              CAMK2N1

              -

              Y↓

              -

              -

              Y↑

              Y↑

              -

              -

              PLCB4

              PM

              -

              -

              -

              -

              -

              -

              -

              CCNH

              -

              -

              -

              -

              -

              -

              -

              -

              PPP2CB

              -

              -

              -

              Y↓

              -

              -

              -

              -

              CCT2

              -

              -

              -

              -

              -

              -

              -

              -

              PPP2R1A

              -

              -

              -

              -

              -

              -

              -

              -

              CD151

              PM

              -

              -

              -

              -

              Y↑

              Y↑

              -

              PSMA7

              -

              -

              -

              Y↓

              -

              -

              -

              -

              COMT

              -

              -

              -

              -

              -

              Y↓

              -

              -

              PTGFR

              PM

              -

              -

              -

              -

              -

              -

              -

              CUEDC2

              -

              -

              -

              -

              -

              -

              -

              -

              QKI

              -

              -

              -

              -

              -

              -

              -

              -

              CXCR7

              PM

              Y↓

              -

              -

              -

              -

              Y↑

              Y↑

              RAMP1

              PM

              -

              Y

              Y↑

              -

              -

              -

              -

              DHRS7

              PM

              -

              -

              -

              -

              -

              Y↓

              -

              RNF208

              -

              -

              -

              -

              -

              -

              -

              -

              EEF1A2

              -

              Y↑

              -

              Y↑

              -

              -

              -

              -

              RPL11

              -

              -

              -

              -

              -

              -

              Y↓

              -

              EEF2

              -

              -

              -

              -

              -

              -

              -

              -

              RPL28

              -

              -

              -

              -

              -

              -

              -

              -

              ELOVL5

              PM

              Y

              Y

              Y↓

              -

              -

              -

              -

              RPS11

              -

              -

              -

              -

              -

              -

              Y↓

              -

              ENDOD1

              S

              Y↑

              -

              -

              -

              -

              -

              -

              RPS18

              -

              -

              -

              Y↑

              -

              -

              -

              -

              ENO2

              PM

              -

              -

              -

              -

              -

              -

              -

              RPS3

              -

              -

              -

              -

              -

              -

              -

              -

              ENSG00000210082

              -

              -

              -

              -

              -

              -

              -

              -

              S100A10

              PM

              -

              -

              -

              -

              -

              -

              -

              ENSG00000211459

              -

              -

              -

              -

              -

              -

              -

              -

              SBDS

              -

              -

              -

              -

              -

              Y↑

              -

              -

              FGFRL1

              PM

              -

              -

              -

              -

              -

              -

              -

              SELENBP1

              -

              Y↓

              -

              Y↓

              -

              -

              -

              -

              FKBP10

              -

              -

              -

              -

              -

              -

              -

              -

              SERINC5

              -

              -

              -

              -

              -

              -

              -

              -

              GALNT3

              -

              -

              -

              Y↑

              -

              Y↓

              -

              -

              SF3A2

              -

              -

              -

              -

              -

              -

              Y↑

              -

              GAS5

              -

              -

              -

              -

              -

              -

              -

              -

              SFRS2B

              -

              -

              -

              -

              -

              -

              -

              -

              GLO1

              -

              -

              -

              Y↑

              Y↑

              Y↑

              -

              -

              SH3BP5L

              -

              -

              -

              -

              -

              -

              -

              -

              GNB2L1

              PM

              -

              -

              -

              -

              -

              Y↑

              -

              SLC25A4

              -

              -

              -

              Y↑

              -

              -

              -

              -

              GRB10

              PM

              -

              -

              -

              -

              -

              -

              -

              SLC25A6

              -

              -

              -

              Y↑

              -

              -

              -

              -

              H2AFJ

              -

              -

              -

              -

              -

              -

              -

              -

              SNX3

              -

              -

              -

              -

              Y↑

              -

              -

              -

              HES6

              -

              -

              -

              -

              -

              -

              Y↑

              Y↑

              SPIRE1

              -

              -

              -

              -

              -

              -

              -

              -

              HLA-B

              PM

              -

              -

              -

              -

              -

              -

              -

              SPON2

              S

              -

              Y

              Y↑

              -

              -

              -

              -

              HMGB2

              -

              -

              -

              -

              -

              -

              -

              Y↑

              STEAP1

              PM

              -

              Y

              Y↑

              -

              -

              -

              -

              HN1

              -

              -

              -

              -

              -

              -

              Y↑

              -

              SYNC1

              -

              -

              -

              -

              -

              -

              -

              -

              HSD17B4

              -

              Y↑

              -

              Y↑

              -

              -

              -

              -

              TFPI

              S

              -

              -

              -

              -

              -

              -

              -

              LOC644844

              -

              -

              -

              -

              -

              -

              -

              -

              TK1

              -

              -

              -

              -

              -

              -

              Y↑

              -

              MAOA

              -

              Y

              -

              -

              Y↑

              -

              -

              -

              TKT

              -

              -

              -

              -

              -

              -

              -

              -

              MARCKSL1

              PM

              -

              -

              Y↑

              -

              -

              -

              -

              TMEM30A

              S&PM

              -

              -

              -

              -

              Y↑

              -

              -

              MDK

              S&PM

              Y↓

              -

              Y↑

              -

              -

              -

              Y↑

              TMEM66

              S&PM

              Y↑

              -

              -

              -

              -

              -

              -

              MT-CO3

              -

              -

              -

              -

              -

              -

              -

              -

              TPD52

              -

              Y↑

              Y

              Y↑

              -

              Y↑

              Y↓

              -

              MT-ND3

              -

              -

              -

              -

              -

              -

              -

              -

              TRPM8

              PM

              Y↑

              -

              Y↑

              -

              -

              -

              Y↓

              NAAA

              -

              -

              -

              -

              -

              -

              -

              Y↑

              UTX

              -

              -

              -

              -

              -

              -

              -

              -

              NAT14

              PM

              -

              -

              -

              -

              -

              -

              -

              VPS13B

              PM

              -

              -

              -

              -

              -

              Y↑

              -

              NELF

              PM

              -

              -

              -

              -

              -

              -

              -

              WDR45L

              -

              -

              -

              -

              -

              -

              -

              -

              NGFRAP1

              -

              -

              -

              Y↓

              -

              -

              -

              -

              YWHAQ

              -

              -

              -

              -

              -

              -

              -

              -

              * Human Genome Nomenclature Committee (HGNC)-approved gene names were used when possible. Non-HGNC-approved gene names were not italicized.

              † S or PM, gene product is thought to be secreted (S) or localize to the plasma membrane (PM)

              ‡ Reg. by A, gene expression changes in response to androgen in prostate cells

              § Spec. to P, gene expression is specific to- or enriched in- prostate tissue compared to other tissues

              II CaP, gene is differentially expressed in prostate cancer compared to normal, benign prostatic hyperplasia, or prostatic intraepithelial neoplasia

              ¶ GG, gene is differentially expressed in higher Gleason grade tissue versus lower Gleason grade tissue

              ** Prog., gene expression correlates with late-stage prostate cancer or is a risk factor that predicts progression

              †† Mets, gene expression is associated with prostate cancer metastasis in human samples or in vivo models

              ‡‡ CR, gene is associated with castration-recurrent prostate cancer in human tissue or in vivo models, but exhibits an opposite trend of this report

              §§ Y, yes; ↑, high gene expression; ↓, low gene expression

              Novel CR-associated genes identify both clinical samples of CRPC and clinical metastasis of prostate cancer

              The expression of novel CR-associated genes were validated in publically available, independent sample sets representing different stages of prostate cancer progression (Gene Expression Omnibus accession numbers: GDS1390 and GDS1439). Dataset GDS1390 includes expression data of ten AS prostate tissues, and ten CRPC tissues from Affymetrix U133A arrays [47]. Dataset GDS1439 includes expression data of six benign prostate tissues, seven localized prostate cancer tissues, and seven metastatic prostate cancer tissues from Affymetrix U133 2.0 arrays [97].

              Unsupervised principal component analysis based on the largest three principal components revealed separate clustering of tumor samples representing AS and CR stages of cancer progression, with the exception of two CR samples and one AS sample (Figure 4a).
              http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-3-43/MediaObjects/12920_2010_Article_178_Fig4_HTML.jpg
              Figure 4

              Principle component analyses of clinical samples. A, Principle component analysis based on the expression of novel CR-associated genes in the downloaded dataset GDS1390 clustered the AS and CR clinical samples into two groups. B, Principle component analysis based on the expression of novel CR-associated genes in the downloaded dataset GDS1439 clustered the clinical samples (benign prostate tissue, benign; localized prostate cancer, Loc CaP; and metastatic prostate cancer, Met CaP) into three groups.

              Metastatic prostate cancer is expected to have a more progressive phenotype and is associated with hormonal progression. Therefore, the gene expression signature obtained from the study of hormonal progression may be common to that observed in clinical metastases. Unsupervised principal component analysis based on the largest three principal components revealed separate clustering of not only benign and malignant, but also localized and metastatic tissue samples (Figure 4b).

              Discussion

              Genes that change levels of expression during hormonal progression may be indicative of the mechanisms involved in CRPC. Here we provide the most comprehensive gene expression analysis to date of prostate cancer with approximately 3 million long tags sequenced using in vivo samples of biological replicates at various stages of hormonal progression to improve over the previous libraries that are approximately 70,000 short tags or less. Previous large-scale gene expression analyses have been performed with tissue samples from men with advanced prostate cancer [4258], and animal or xenograft models of CRPC [5969]. Most of these previous studies compared differential expression between CRPC samples with the primary samples obtained before androgen ablation. This experimental design cannot distinguish changes in gene expression that are a direct response to androgen ablation, or from changes in proliferation/survival that have been obtained as the prostate cancer cells progress to more a more advanced phenotype. Here we are the first to apply an in vivo model of hormonal progression to compare gene expression between serial samples of prostate cancer before (AS), and after androgen ablation therapy (RAD) as well as when the cells become CR. This model is the LNCaP Hollow Fiber model [21] which has genomic similarity with clinical prostate cancer [23] and mimics the hormonal progression observed clinically in response to host castration as measured by levels of expression of PSA and cell proliferation. Immediately prior to castration, when the cells are AS, PSA levels are elevated and the LNCaP cells proliferate. A few days following castration, when the cells are RAD, PSA levels drop and the LNCaP cells cease to proliferate, but do not apoptose in this model. Approximately 10 weeks following castration, when the cells are CR, PSA levels rise and the LNCaP cells proliferate in the absence of androgen. This model overcomes some limitations in other studies using xenografts that include host contamination of prostate cancer cells. The hollow fibers prevent infiltration of host cells into the fiber thereby allowing retrieval of pure populations of prostate cells from within the fiber. The other important benefit of the fiber model is the ability to examine progression of cells to CRPC at various stages within the same host mouse over time, because the retrieval of a subset of fibers entails only minor surgery. The power to evaluate progression using serial samples from the same mouse minimizes biological variation to enhance the gene expression analyses. However, limitations of this model include the lack of cell-cell contact with stroma cells, and lack of heterogeneity in tumors. Typically, these features would allow paracrine interactions as expected in clinical situations. Consistent with the reported clinical relevance of this model [23], here principal component analysis based on the expression of these novel genes identified by LongSAGE, clustered the clinical samples of CRPC separately from the androgen-dependent samples. Principal component analysis based on the expression of these genes also revealed separate clustering of the different stages of tumor samples and also showed separate clustering of the benign samples from the prostate cancer samples. Therefore, some common changes in gene expression profile may lead to the survival and proliferation of prostate cancer and contribute to both distant metastasis and hormonal progression. We used this LNCaP atlas to identify changes in gene expression that may provide clues of underlying mechanisms resulting in CRPC. Suggested models of CRPC involve: the AR; steroid synthesis and metabolism; neuroendocrine prostate cancer cells; and/or an imbalance of cell growth and cell death.

              Androgen receptor (AR)

              Transcriptional activity of AR

              The AR is suspected to continue to play an important role in the hormonal progression of prostate cancer. The AR is a ligand-activated transcription factor with its activity altered by changes in its level of expression or by interactions with other proteins. Here, we identified changes in expression of some known or suspected modifier of transcriptional activity of the ARin CRPC versus RAD such as Cyclin H (CCNH) [107], proteasome macropain subunit alpha type 7 (PSMA7) [108], CUE-domain-containing-2 (CUEDC2) [109], filamin A (FLNA) [110], and high mobility group box 2 (HMGB2) [111]. CCNH and PSMA7 displayed increased levels of expression, while CUEDC2, FLNA, and HMGB2 displayed decreased levels of expression in CR. The expression trends of CCNH, CUEDC2, FLNA, and PSMA7 in CRPC may result in increased AR signaling through mechanisms involving protein-protein interactions or altering levels of expression of AR. CCNH protein is a component of the cyclin-dependent activating kinase (CAK). CAK interacts with the AR and increases its transcriptional activity [107]. Over-expression of the proteosome subunit PSMA7 promotes AR transactivation of a PSA-luciferase reporter [108]. A fragment of the protein product of FLNA negatively regulates transcription by AR through a physical interaction with the hinge region [110]. CUEDC2 protein promotes the degradation of progesterone and estrogen receptors [109]. These steroid receptors are highly related to the AR, indicating a possible role for CUEDC2 in AR degradation. Thus decreased expression of FLNA or CUEDC2 could result in increased activity of the AR. Decreased expression of HMGB2 in CRPC is predicted to decrease expression of at least a subset of androgen-regulated genes that contain palindromic AREs [111]. Here, genes known to be regulated by androgen were enriched in expression trend categories with a peak or valley at the RAD stage of prostate cancer progression. Specifically, 8 of the 13 tags (62%) exhibiting these expression trends 'E', 'F', 'J', 'K', or 'L' represented known androgen-regulated genes, in contrast to only 22 of the remaining 122 tags (18%; Tables 4 &5). Overall, this data supports increased AR activity in CRPC, which is consistent with re-expression of androgen-regulated genes as previously reported [68] and similarity of expression of androgen regulated genes between CRPC and prostate cancer before androgen ablation [23].

              Steroid synthesis and metabolism

              In addition to changes in expression of AR or interacting proteins altering the transcriptional activity of the AR, recent suggestion of sufficient levels of residual androgen in CRPC provides support for an active ligand-bound receptor [112]. The AR may become re-activated in CRPC due to the presence of androgen that may be synthesized by the prostate de novo [4] or through the conversion of adrenal androgens. Here, the expression of 5 genes known to function in steroid synthesis or metabolism were significantly differentially expressed in CRPC versus RAD. They are 24-dehydrocholesterol reductase (DHCR24) [113], dehydrogenase/reductase SDR-family member 7 (DHRS7) [114], elongation of long chain fatty acids family member 5 (ELOVL5) [115, 116], hydroxysteroid (17-beta) dehydrogenase 4 (HSD17B4) [117], and opioid receptor kappa 1 (OPRK1) [118]. Increased levels of expression of these genes may be indicative of the influence of adrenal androgens, or the local synthesis of androgen, to reactivate the AR to promote the progression of prostate cancer in the absence of testicular androgens.

              Neuroendocrine

              Androgen-deprivation induces neuroendocrine differentiation of prostate cancer. Here, the expression of 8 genes that are associated with neuroendocrine cells were significantly differentially expressed in CRPC versus RAD. They either responded to androgen ablation such as hairy and enhancer of split 6 (HES6) [119], karyopherin/importin beta 1 (KPNB1) [120], monoamine oxidase A (MAOA)[121], and receptor (calcitonin) activity modifying protein 1 (RAMP1) [122]], or were increased expressed in CRPC such as ENO2 [122], OPRK1 [118], S100 calcium binding protein A10 (S100A10) [123], and transient receptor potential cation channel subfamily M member 8 (TRPM8) [124].

              Proliferation and Cell survival

              The gene expression trends of GAS5 [125], GNB2L1 [126], MT-ND3, NKX3-1 [127], PCGEM1 [128], PTGFR [129], STEAP1 [130], and TMEM30A [131] were in agreement with the presence of proliferating cells in CRPC. Of particular interest is that we observed a transcript anti-sense to NKX3-1, a tumor suppressor, highly expressed in the stages of cancer progression that were AS and CR, but not RAD. Anti-sense transcription may hinder gene expression from the opposing strand, and therefore, represents a novel mechanism by which NKX3-1 expression may be silenced. There were also some inconsistencies including the expression trends of BTG1 [132], FGFRL1 [133], and PCOTH [134] and that may be associated with non-cycling cells. Overall, there was more support at the transcriptome level for proliferation than not, which was consistent with increased proliferation observed in the LNCaP Hollow Fiber model [21].

              Gene expression trends of GLO1 [135], S100A10 [136], TRPM8 [137], and PI3KCD [138] suggest cell survival pathways are active following androgen-deprivation and/or in CRPC, while gene expression trends of CAMK2N1 [139], CCT2 [140], MDK [141, 142], TMEM66 [143], and YWHAQ [136] may oppose such suggestion. Taken together, these data neither agree nor disagree with the activation of survival pathways in CRPC. In contrast to earlier reports in which MDK gene and protein expression was determined to be higher in late stage cancer [63, 142], we observed a drop in the levels of MDK mRNA in CRPC versus RAD. MDK expression is negatively regulated by androgen [65]. Therefore, the decreased levels of MDK mRNA in CRPC may suggest that the AR is reactivated in CRPC.

              Other

              The significance of the gene expression trends of AMD1, BNIP3, GRB10, MARCKSL1, NGRAP1, ODC1, PPP2CB, PPP2R1A, SLC25A4, SLC25A6, and WDR45L that function in cell growth or cell death/survival were not straightforward. For example, BNIP3 and WDR45L, both relatively highly expressed in CRPC versus RAD, may be associated with autophagy. BNIP3 promotes autophagy in response to hypoxia [144], and the WDR45L-related protein, WIPI-49, co-localizes with the autophagic marker LC3 following amino acid depletion in autophagosomes [145]. It is not known if BNIP3 or putative WDR45L-associated autophagy results in cell survival or death. Levels of expression of NGFRAP1 were increased in CRPC versus RAD. The protein product of NGFRAP1 interacts with p75 (NTR). Together they process caspase 2 and caspase 3 to active forms, and promote apoptosis in 293T cells [146]. NGFRAP1 requires p75 (NTR) to induce apoptosis. However, LNCaP cells do not express p75 (NTR), and so it is not clear if apoptosis would occur in this cell line [147].

              Overall, genes involved in cell growth and cell death pathways were altered in CRPC. Increased tumor burden may develop from a small tip in the balance when cell growth outweighs cell death. Unfortunately, the contributing weight of each gene is not known, making predictions difficult based on gene expression alone of whether proliferation and survival were represented more than cell death in this model of CRPC. It should be noted that LNCaP cells are androgen-sensitive and do not undergo apoptosis in the absence of androgens. The proliferation of these cells tends to decrease in androgen-deprived conditions, but eventually with progression begins to grow again mimicking clinical CRPC.

              Conclusion

              Here, we describe the LNCaP atlas, a compilation of LongSAGE libraries that catalogue the transcriptome of human prostate cancer cells as they progress to CRPC in vivo. Using the LNCaP atlas, we identified differential expression of 96 genes that were associated with castration-recurrence in vivo. These changes in gene expression were consistent with the suggested model for a role of the AR, steroid synthesis and metabolism, neuroendocrine cells, and increased proliferation in CRPC.

              Author's information

              M.D.S. and M.A.M. are Terry Fox Young Investigators. M.A.M. is a Senior Scholar of the Michael Smith Foundation for Health Research.

              Abbreviations

              ACPP

              prostate acid phosphatise

              ACTH: 

              adrenocorticotropic hormone

              AR: 

              androgen receptor

              AREs: 

              androgen response elements

              AS: 

              androgen-sensitive

              BAX

              BCL2-associated X protein

              BCL-2

              B-cell CLL/lymphoma 2

              BCL2L1

              BCL2-like 1

              CAK: 

              cyclin-dependent activating kinase

              CCND1

              cyclin D1

              CCNH: 

              Cyclin H

              CDKN1A

              cyclin-dependent kinase inhibitor 1A

              CDKN1B

              cyclin-dependent kinase inhibitor 1B

              CHG

              chromogranin

              CR: 

              castration-recurrent

              CRPC: 

              castration-recurrent prostate cancer

              CUEDC2: 

              CUE-domain-containing-2

              DHCR24: 

              24-dehydrocholesterol reductase

              DHRS7: 

              dehydrogenase/reductase SDR-family member 7

              EASE: 

              Expression Analysis Systematic Explorer

              ELOVL5: 

              elongation of long chain fatty acids family member 5

              ENO2: 

              neuronal enolase 2

              FLNA: 

              filamin A

              GO: 

              Gene Ontology

              HES6: 

              hairy and enhancer of split 6

              HGNC: 

              HUGO Gene Nomenclature Committee

              HMGB2: 

              high mobility group box 2

              HMGCS1

              3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1

              HPA: 

              hypothalamus-pituitary-adrenal

              HSD17B3

              hydroxysteroid (17-beta) dehydrogenase 3

              HSD17B4: 

              hydroxysteroid (17-beta) dehydrogenase 4

              HSD17B5

              hydroxysteroid (17-beta) dehydrogenase 5

              IL6: 

              interleukin 6

              KEGG: 

              Kyoto Encyclopedia of Genes and Genomes

              KLK3

              kallikrein 3

              KPNB1: 

              karyopherin/importin beta 1

              LHRH: 

              Leutinizing hormone releasing hormone

              LongSAGE: 

              long serial analysis of gene expression

              MAOA: 

              monoamine oxidase A

              NCOA

              nuclear receptor coactivator

              NKX3-1

              NK3 homeobox 1

              NTS

              neurotensin

              OPRK1: 

              opioid receptor kappa 1

              PKA: 

              protein kinase A

              PSA: 

              prostate-specific antigen also known as KLK3

              PSMA7: 

              proteasome macropain subunit alpha type 7

              PTHrP: 

              parathyroid hormone-related protein

              qRT-PCR: 

              quantitative real time-polymerase chain reaction

              RAD: 

              responsive to androgen-deprivation

              RAMP1: 

              receptor (calicitonin) activity modifying protein 1

              RB1

              retinoblastoma 1

              S100A10: 

              S100 calcium binding protein A10

              SQLE

              squalene epoxidase

              TRPM8: 

              transient receptor potential cation channel subfamily M member 8.

              Declarations

              Acknowledgements

              The authors would like to thank Jean Wang for her excellent technical assistance and Dr. Simon Haile for helpful discussions. This work was supported by funding from NIH, Grant CA105304 (M.D.S.).

              Authors’ Affiliations

              (1)
              Genome Sciences Centre, British Columbia Cancer Agency

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