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. 2013:2013:763569.
doi: 10.1155/2013/763569. Epub 2013 Jan 10.

Androgen receptor-_target genes in african american prostate cancer disparities

Bi-Dar Wang  1 , Qi Yang, Kristin Ceniccola, Fernando Bianco, Ramez Andrawis, Thomas Jarrett, Harold Frazier, Steven R Patierno, Norman H Lee
Affiliations

Androgen receptor-_target genes in african american prostate cancer disparities

Bi-Dar Wang et al. Prostate Cancer. 2013.

Abstract

The incidence and mortality rates of prostate cancer (PCa) are higher in African American (AA) compared to Caucasian American (CA) men. To elucidate the molecular mechanisms underlying PCa disparities, we employed an integrative approach combining gene expression profiling and pathway and promoter analyses to investigate differential transcriptomes and deregulated signaling pathways in AA versus CA cancers. A comparison of AA and CA PCa specimens identified 1,188 differentially expressed genes. Interestingly, these transcriptional differences were overrepresented in signaling pathways that converged on the androgen receptor (AR), suggesting that the AR may be a unifying oncogenic theme in AA PCa. Gene promoter analysis revealed that 382 out of 1,188 genes contained cis-acting AR-binding sequences. Chromatin immunoprecipitation confirmed STAT1, RHOA, ITGB5, MAPKAPK2, CSNK2A,1 and PIK3CB genes as novel AR _targets in PCa disparities. Moreover, functional screens revealed that androgen-stimulated AR binding and upregulation of RHOA, ITGB5, and PIK3CB genes were associated with increased invasive activity of AA PCa cells, as siRNA-mediated knockdown of each gene caused a loss of androgen-stimulated invasion. In summation, our findings demonstrate that transcriptional changes have preferentially occurred in multiple signaling pathways converging ("transcriptional convergence") on AR signaling, thereby contributing to AR-_target gene activation and PCa aggressiveness in AAs.

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Figures

Figure 1
Figure 1
Predicted AR-_target genes in AA PCa are overrepresented in cancer-associated signaling pathways. Flow chart outlining the strategy of combining gene expression profiling comparing 20 AA PCa specimens with 15 CA PCa specimens, gene promoter analysis, and pathway analysis to identify direct AR-_target genes associated with PCa disparities. A total of 1,188 significant differentially express genes (ANOVA with 10% FDR) were subjected to hierarchical clustering analysis (clustering diagram, upper left, highly expressed genes in red, weakly expressed genes in blue) and pathway analysis by IPA (representative canonical pathways, upper right). Among the 1,188 significant genes, 382 genes were predicted as AR-_target/responsive genes using the ALGGEN-PROMO program. These 382 genes were again subjected to clustering and pathway analysis to identify significant AR-associated pathways in AA PCa.
Figure 2
Figure 2
Quantitative RT-PCR validation of microarray results in patient specimens and population-specific PCa cell lines. qRT-PCR experiments were conducted to compare the gene expression levels of GIT1, STAT1, EIF3B, FGF13, RHOA, ITGB5, MAPKAPK2, STAT2, RHOU, CSNK2A1, and PIK3CB in (a) PCa specimens derived from AA and CA patients, (b) AA PCa cell line MDA PCa 2b (2b) versus CA PCa cell line VCaP, and (c) AA PCa cell line E006AA versus CA PCa cell line VCaP. The relative gene expression level in patient specimens (a) was determined by 2−ΔCt using EIF1AX as the control gene for normalization. Box-and-Whisker and dot plots represent the average and individual values, respectively, of gene expression in the tested tissue samples. The log2 ratio values in (b) and (c) were determined by subtracting the log2 signal intensity of AA cancer with the log2 signal intensity of CA cancer for each gene from microarray results. For qRT-PCR results from cell line comparisons in (b) and (c), log⁡2 ratio values were calculated by the ΔΔCt method using EIF1AX as the control gene for normalization [35]. Data are represented as the mean ± SEM of 20 AA PCa and 15 CA PCa samples for microarray experiments and 3–5 independent PCa cell line experiments. *Significantly different between AA PCa versus CA PCa from microarray results (ANOVA, 10% FDR). **Significantly different between AA versus CA PCa cell lines from qRT-PCR results (P < 0.05, Student's t-test).
Figure 3
Figure 3
Enrichment of AR binding to _target genes in AA PCa cells compared to CA PCa cells. (a) Representative ChIP-PCR assays in the AA PCa cell line E006AA and CA PCa cell line VCaP. ChIP DNA from AR-immunoprecipitates (AR), no antibody control (no Ab) or starting chromatin DNA (Input) was amplified using PCR with primers specific to predicted AR binding sites in the promoter regions of STAT1, RHOA, ITGB5, MAPKAPK2, CSNK2A1, and PIK3CB genes. KLK3 (PSA gene) and ACTB were used as positive and negative controls for the ChIP-PCR assays, respectively. (b) Quantification of AR enrichment on _target genes in AA and CA PCa cell lines. The AR occupancies on the _target genes were measured based on the percentages of ChIP-to-Input signals [% Input = (ChIP signal/Input signal)/dilution rate × 100%]. Quantification of ChIP and Input signals were calculated using the Image J program [37] from NIH. Data are represented as the mean ± SD (standard deviation) of 3–5 independent ChIP and PCR experiments. *Significantly different AR occupancies at AA versus CA _target genes (P < 0.05 using Student's t-test).
Figure 4
Figure 4
Androgen stimulation increases AR occupancy at _target genes and upregulates AR-_target gene expression in AA PCa cells. (a) AA PCa cell line MDA PCa 2b and (b) AA PCa cell line E006AA were treated with 100 nM DHT for 18 hr (top panels). Representative ChIP-PCR assays confirmed DHT-induced increases in AR occupancies at the promoter regions of RHOA, ITGB5, and PIK3CB genes. AR occupancies and relative gene expressions with or without androgen stimulation (bottom panels) were measured as a percentage of ChIP-to-Input signal and mRNA levels, respectively. KLK3 (PSA gene) and ACTB were used as positive and negative controls for the ChIP-PCR assays, respectively. For qRT-PCR, the relative expression levels of RHOA, ITGB5, PIK3CB, and KLK3 were determined by the ΔΔCt method using EIF1AX and PPA1 as endogenous genes for normalization. Cells were treated with a vehicle (<0.01% ethanol final concentration) or DHT for 18 hr prior to ChIP and qRT-PCR assays. Data are represented as the mean ± SEM of 3–5 independent ChIP and PCR experiments. *Significantly different AR occupancies and expression levels of AR-_target genes in both cell lines (P < 0.05 using Student's t-test).
Figure 5
Figure 5
AR-_target genes promote cell invasion in an androgen-dependent manner in AA cancer cells. (a) Androgen stimulation (10 nM DHT for 48 hr) increases invasion in the AA PCa cell lines MDA PCa 2b and E006AA. (b) siRNA-mediated knockdown of AR-_target genes RHOA, ITGB5, or PIK3CD diminished androgen-induced (10 nM DHT for 48 hr) cell invasion in both MDA PCa 2b and E006AA cell lines. Knockdown efficiency (determined for each experiment) was typically ~80% as determined by qRT-PCR (data not shown). Data are represented as the mean ± SEM of 4–7 independent cell invasion assays. *Significant difference between cells treated with vehicle (<0.01% ethanol final concentration) versus 10 nM DHT using an ANOVA with Holm post hoc test (P < 0.05). **Significant difference between vehicle-treated cells incubated with siRNA against RHOA, ITGB5, or PIK3CB versus vehicle-treated cells incubated with nonsense siRNA (siNS) using an ANOVA with Holm post hoc test (P < 0.05). ***Significant difference between DHT-stimulated cells incubated with siRNA against RHOA, ITGB5, or PIK3CB versus DHT-stimulated cells incubated with nonsense siRNA (siNS) using an ANOVA with Holm post hoc test (P < 0.05).
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