An Sp1 Modulated Regulatory Region Unique to Higher Primates Regulates Human Androgen Receptor Promoter Activity in Prostate Cancer Cells

Abstract

Androgen receptor (AR) mediated signalling is necessary for normal development of the prostate gland and also drives prostate cancer (PCa) cell growth and survival, with many studies showing a correlation between increased receptor levels and therapy resistance with progression to fatal castrate recurrent PCa (CRPC). Although it has been held for some time that the transcription factor Sp1 is the main stimulator of AR gene transcription, comprehensive knowledge of the regulation of the AR gene remains incomplete. Here we describe and characterise in detail two novel active regulatory elements in the 5'UTR of the human AR gene. Both of these elements contain overlapping binding sites for the positive transcription factor Sp1 and the repressor protein pur-α. Aberrant cell signalling is characteristic of PCa and the transcriptional activity of the AR promoter in PCa cells is dependent upon the relative amounts of the two transcription factors. Together with our corroboration of the dominant role of Sp1, the findings support the rationale of targeting this transcription factor to inhibit tumour progression. This should be of particular therapeutic relevance in CRPC where the levels of the repressor pur-α are reduced.

Fig 1. Regulatory elements in human AR 5’UTR.

(A) Diagrammatic representation of the human AR gene 5’UTR and immediate proximal promoter showing the principal regulatory elements and the regions under study. Bent arrow indicates the transcriptional start site (+1) and ATG with solid arrow show the start of translation. (B) Potential GC box (green dashed box) within the confirmed human ARS regulatory element (blue underlined). (C) Alignment of the human AR 5’UTR and the mouse AR 5’UTR suppressor element protected from DNase I digestion [40] (blue underlined) with the confirmed human GC boxes (green solid box). Homologous sequences are indicated by vertical lines.

Fig 2. Multiple genome alignments of the AR 5’UTR.

The regions of the hAR gene 5’UTR under investigation were compared to those of the indicated placental species. (A) the ARS (blue box), and (B) the Sp1-3 regulatory element (green box). Differences from the human sequence are indicated by bold, underlined font.

Fig 3. Effect of mithramycin A on AR mRNA and protein expression.

LNCaP cells were incubated with mithramycin A for 24 h and then assayed. (A) Western blot analysis with the relative values of AR/GAPDH shown below. (B) RT-PCR of AR and GAPDH mRNA. LNCaP cells were transfected with phAR1.6Luc (WT) or phAR1.6Luc-ΔCG and treated with DMSO or 50 nM mithramycin A for 24 hours and luciferase activity was measured. Data represent the means ± SD of at least three independent experiments and the statistical significance of the indicated comparisons are: * p<0.05; ** p<0.01 and *** p<0.001.

Fig 4. Effect of mutations on transcriptional activity.

(A) Schematic diagram of the luciferase reporter construct phAR1.6Luc driven by 1.6 kbp of the hAR promoter and 5’UTR with potential transcription factor binding to the regions of interest. Sp1-3 contains two Sp1 binding sites. (B and C) The indicated PCa cell lines, cultured in complete medium, were transfected with either phAR1.6Luc containing the WT sequence (black bars) or the mutated version (grey bars) shown above each chart. Luciferase data represent the means ± SEM of at least three independent experiments and the statistical significance of the indicated comparisons are: * p<0.05; ** p<0.01 and *** p<0.001.

Fig 5. Binding of Sp1 to the AR gene promoter and 5’UTR sequences.

Purified Sp1 protein (panel A) or nuclear extract from DU145 cells (panels (B) to (D)) were incubated with the labelled oligonucleotide probes indicated below each gel and the products resolved by electrophoretic mobility shift analysis. Additions are shown above the gels and were: Ab, antibody; PI, pre-immune serum; Mith, mithramycin; Sp1 cons, consensus Sp1 oligonucleotide. Competing unlabelled oligonucleotides (100 fold molar excess) or immune sera were added prior to addition of probe. EMSAs are representative of at least 3 independent experiments. (A) The indicated labelled probes were incubated with purified Sp1 protein except in lane 1 which had no addition. Black arrow indicates Sp1-DNA complex. (B) Anti-Sp1 antibody was added as indicated, and the complex absent after incubation with antibody is indicated by the arrow. (C) Mithramycin (120 nM) was added to indicated lanes and the complex depleted after incubation with the drug is indicated by the solid arrow. (D) Unlabelled competing oligonucleotide encoding Sp1 consensus binding site was added as shown.

Fig 6. The AR 5’UTR possesses overlapping regulatory elements.

Nuclear extract from DU145 cells were incubated with the labelled oligonucleotide probes indicated below each gel and the products resolved by electrophoretic mobility shift analysis. Additions are shown above the gels and were: Ab, antibody; PI, pre-immune serum. EMSAs are representative of at least 3 independent experiments. (A) Either double stranded probes or single stranded oligonucleotides of the reverse strand were incubated with nuclear extract and protein-DNA complexes resolved: black arrow indicates Sp1 and open arrow indicates pur-α binding respectively. (B) Forward and reverse single stranded labelled oligonucleotides of the regulatory elements were incubated with nuclear extract. Anti-hnRNP-K antibody was included where indicated and hnRNP-K-DNA complexes indicated by grey arrow and open arrow indicates pur-α binding.

Fig 7. ChIP analysis confirms binding of Sp1 to 5’UTR ARS.

(A) Line diagram (not to scale) of the hAR 5’UTR showing the recognition sites of the restriction endonuclease HphI used to digest plasmid, plus the forward (F) and reverse (R) primers (solid arrows) used for ChIP semi-quantitative PCR. Oligonucleotide Vect-R was specific for the plasmid vector sequences, and the bent arrow indicates the transcriptional start site. (B). Representative agarose gels of PCR amplified DNA immunoprecipitated with anti-Sp1 antibody in which the order of some lanes have been altered to aid clarity and facilitate comparisons; IP, input sample; Ig, pre-immune rabbit IgG; Ab, anti-Sp1 antibody.

Fig 8. Variation of transcription factor levels in PCa cells.

Western blot analysis of Sp1, pur-α and hnRNP-K protein levels normalised against GAPDH in the indicated PCa cell lines. The data represent the means ± SEM of at least three independent experiments and statistical significance is: *** p<0.001.

Fig 9. Model for regulation of the human AR gene.

The organisation of positive (GC-box) and negative (ARS, nARE) elements in the promoter and 5’UTR of the AR gene are shown together with the relevant transcription factors.