Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements

Journal name:
Nature Genetics
Volume:
42,
Pages:
668–675
Year published:
DOI:
doi:10.1038/ng.613
Received
Accepted
Published online
Corrected online

Abstract

DNA double-strand breaks (DSBs) can lead to the development of genomic rearrangements, which are hallmarks of cancer. Fusions between TMPRSS2, encoding the transmembrane serine protease isoform 2, and ERG, encoding the v-ets erythroblastosis virus E26 oncogene homolog, are among the most common oncogenic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSBs. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of androgen receptor and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSBs in generating TMPRSS2-ERG rearrangements.

At a glance

Figures

  1. TOP2B is required for efficient induction of androgen receptor target gene expression after androgen stimulation.
    Figure 1: TOP2B is required for efficient induction of androgen receptor target gene expression after androgen stimulation.

    (a) Inhibition of TOP2B with etoposide (ET) or merbarone (Mer) before DHT stimulation of LNCaP cells attenuated expression of an androgen-induced geneset (P < 10−14), defined as the 45 genes upregulated >2-fold in DHT- compared with control-treated LNCaP cells. (b) Geneset enrichment analysis shows highly statistically significant enrichment for downregulation of three independently derived but overlapping androgen-responsive genesets in ET/DHT vs. DHT, Mer/DHT vs. DHT, and sh-TOP2B/DHT vs. DHT treatment groups (see also Supplementary Figs. 2 and 3). y axis shows the degree of confidence for geneset enrichment as −log10(P-value). Significance threshold corresponding to P = 0.05 is indicated. (c,d) Real-time RT-PCR analysis of relative expression of selected genes with respect to GAPDH expression confirms downregulation of representative androgen-responsive genes in ET/DHT, Mer/DHT and sh-TOP2B/DHT-treated cells. Data are shown as mean ± s.e.m. of two to three replicates.

  2. Androgen stimulation induced recruitment of androgen receptor-TOP2B and TOP2B catalytic cleavage at known regulatory regions of androgen receptor target genes.
    Figure 2: Androgen stimulation induced recruitment of androgen receptor–TOP2B and TOP2B catalytic cleavage at known regulatory regions of androgen receptor target genes.

    (a) Schematic of AREs in the promoter and enhancer regions of KLK3 and TMPRSS2. Bars indicate positions (relative to transcriptional start site) of amplicons analyzed. (b) ChIP analysis of androgen-deprived LAPC4 cells that were stimulated with 100 nM DHT for varying time points. DHT induced recruitment of androgen receptor and TOP2B to the enhancers and promoters of KLK3 and TMPRSS2 but not to an intervening region (middle) between the enhancer and promoter of KLK3. (c) Stimulation of androgen-depleted LAPC4 cells with 100 nM DHT for 6 h allows coimmunoprecipitation of TOP2B with androgen receptor antibodies and vice versa. IP, immunoprecipitating antibody; IB, immunoblotting antibody; TCL, total cell lysate. (d) ChIP-re-ChIP experiments show that stimulation of androgen-deprived LAPC4 cells with 100 nM DHT induces co-recruitment of androgen receptor and TOP2B to AREs of KLK3 and TMPRSS2. The first round of ChIP was performed using anti-TOP2B antibodies and the resulting immunoprecipitates were subjected to a second round of ChIP with anti-androgen receptor antibodies. Relative enrichment was determined by quantitative (q)PCR and is shown as percentage of input DNA. (e,f) KSDS method shows TOP2 catalytic cleavage of androgen receptor target sites in LAPC4 cells in a DHT- and TOP2B-dependent fashion. Results are presented as mean percentage of input with ± s.e.m. of two to three replicates.

  3. Androgen stimulation induces androgen receptor-TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human prostate cancer.
    Figure 3: Androgen stimulation induces androgen receptor–TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human prostate cancer.

    (a) Sites closest to TMPRSS2-ERG breakpoints from eight prostate cancer cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (P = 0.010 and 0.013 for TMPRSS2 and ERG breakpoints, respectively) in LAPC4 cells16, 17. Labeled sites (for example, T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. (b) DHT- and TOP2B-dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). (c) SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Figs. 9 and 10 for overview of SLOT). (d) ChIP enrichment of androgen receptor (AR) and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. (e,f) 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± s.e.m. of two to three experiments.

  4. Androgen stimulation results in TOP2B-dependent DSB formation.
    Figure 4: Androgen stimulation results in TOP2B-dependent DSB formation.

    (a) Androgen stimulation induced formation of γH2A.X foci in LAPC4 cells. (b) Androgen stimulation led to recruitment of ATM to representative sites of high TOP2B activity but much less so to a region showing low TOP2B catalytic cleavage (E35). (c,d) Stimulation of LAPC4 cells with DHT induced DSBs that could be end-labeled with biotin at representative TOP2 cleavage sites, but not at regions showing low TOP2 cleavage (KLK3 middle, E35). (e) DSBs largely resolve by 24 h after DHT stimulation of LAPC4 cells as shown by reduced biotin labeling and ATM recruitment. The time course of DSB formation/resolution and ATM recruitment are highly parallel. (f) DHT-induced DSBs at a representative TOP2B enriched site (T8) depend on TOP2B. A site showing low TOP2B enrichment (T23) served as a negative control. (g) DHT-induced strand breaks at representative TMPRSS2 sites are enriched for TOP2B and ATM binding as seen by ChIP-re-ChIP experiments. Results are presented as mean percentage of input with ± s.e.m. of two to three replicates. (h) DHT induces chromosomal breaks at TMPRSS2 in LAPC4 cells, monitored as fraction of cells (n > 200) showing split-apart of FISH probes 5′ (green) and 3′ (red) of TMPRSS2. (i) Treatment of LAPC4 cells with ET ± DHT increased split-aparts at ERG compared to control or DHT treatment. Error bars indicate 95% confidence intervals.

  5. Androgen-induced TOP2B-mediated DSBs are recombinogenic and promote de novo production of TMPRSS2-ERG fusion genes.
    Figure 5: Androgen-induced TOP2B-mediated DSBs are recombinogenic and promote de novo production of TMPRSS2-ERG fusion genes.

    (a) Selection of TMPRSS2 regions showing high (intron 1-T8) and low (TMPRSS2-exon 6) KSDS enrichment in response to DHT stimulation of LAPC4 cells. (b) DHT-induced breaks can be detected in plasmids containing sequences surrounding region T8 of TMPRSS2 intron 1 (pcDNA6.2-IN-1) as shown by increased biotin labeling in DHT-stimulated LAPC4 cells transfected with this plasmid. Plasmids containing TMPRSS2 exon 6 (pcDNA6.2-EX-6) served as a negative control. (c) Schematic of androgen-induced genomic recombination assay in LAPC4 cells transfected with pcDNA6.2-IN-1 or pcDNA6.2-EX-6, which contain a blasticidin-resistance gene. Number of colonies represents the number of recombination events allowing integration of the blasticidin resistance vectors into the LAPC4 genome. (d) Representative results of genomic recombination assays. (e) pcDNA6.2-IN-1–transfected LAPC4 cells produced significantly more androgen-induced recombination events than pcDNA6.2-EX-6–transfected cells. Treatment with sh-TOP2B abolished this effect. (f) Although both showed similar recombination frequency at the vector backbone, recombination frequency in the IN-1 insert was significantly higher than in the EX-6 insert in pooled colonies as determined using the strategy shown on the right. Data are shown as mean ± s.e.m. of two to three replicates. (g) DHT stimulation of LAPC4 cells leads to increased TMPRSS2-ERG fusion transcripts compared to background levels in LAPC4 cells grown in androgen-containing (steady-state) or androgen-deprived (control) media. Pharmacological or genetic modulation of TOP2B (Mer, sh-TOP2B), PARP1 (3-AB, PJ-34, si-PARP1) or DNA-PKCS (Wort, si-DNA-PKCS) reduces TMPRSS2-ERG fusion transcripts without significantly altering GAPDH or TBP expression. (h) DHT stimulation leads to de novo formation of TMPRSS2-ERG fusion transcripts in LNCaP cells.

  6. TMPRSS2-ERG rearrangements are observed in PIN prostate cancer precursor lesions and are associated with changes in TOP2B expression.
    Figure 6: TMPRSS2-ERG rearrangements are observed in PIN prostate cancer precursor lesions and are associated with changes in TOP2B expression.

    (a) H&E stain and corresponding TMPRSS2 FISH in a representative PIN lesion. FISH image of red boxed area shows interphase nuclei of PIN lesion with split-aparts of 5′ (green arrow) and 3′ (red arrow) TMPRSS2 FISH probes. (b) H&E stain and corresponding TMPRSS2 FISH (green boxed area) of normal prostate epithelium. Orange arrows indicate normal configuration of 5′ and 3′ TMPRSS2 FISH probes. Scale bars, 10 μm. (c) Normal prostate epithelium shows strong expression of TOP2B in basal cells (red arrow) and low expression in luminal cells, whereas androgen receptor is predominantly expressed in luminal cells (green arrow). Lumen of prostate gland is marked with an asterisk. (d) PIN lesion shows high TOP2B in basal and luminal cells (purple arrow). Note the accentuated nucleolar localization of TOP2B (white arrow).

  7. Proposed model for androgen-induced TOP2B-mediated double strand breaks and TOP2B instability (TIN) for the formation of TMPRSS2-ERG gene fusions.
    Figure 7: Proposed model for androgen-induced TOP2B-mediated double strand breaks and TOP2B instability (TIN) for the formation of TMPRSS2-ERG gene fusions.

    Androgen stimulation leads to co-recruitment of ligand-bound androgen receptor and TOP2B to regulatory regions of androgen-responsive genes as well as to regions of TMPRSS2 and ERG genes that participate in genomic rearrangements. TOP2B catalytic activity and the associated DSB formation are required for efficient activation of androgen-responsive genes, and these DSBs are usually resolved shortly after induction. In some circumstances, stabilization of these TOP2B-mediated DSBs can lead to illegitimate recombination and rare rearrangements between TMPRSS2 or other androgen-responsive genes and ERG that are then subject to selection during neoplastic outgrowth.

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Change history

Corrected online 18 July 2010
In the version of this article initially published online, there were four sentences (in the Results on pages 2 and 3, in the legend to Figure 3 and in the Online Methods) containing minor errors. These errors have been corrected for the print, PDF and HTML versions of this article.

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Author information

  1. These authors contributed equally to this work.

    • Martin J Aryee &
    • Antoun Toubaji

Affiliations

  1. Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA.

    • Michael C Haffner,
    • Martin J Aryee,
    • David M Esopi,
    • William B Isaacs,
    • Alan K Meeker,
    • George Netto,
    • Angelo M De Marzo,
    • William G Nelson &
    • Srinivasan Yegnasubramanian
  2. Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.

    • Martin J Aryee
  3. Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.

    • Antoun Toubaji,
    • Roula Albadine,
    • Bora Gurel,
    • G Steven Bova,
    • Alan K Meeker,
    • George Netto,
    • Angelo M De Marzo &
    • William G Nelson
  4. Brady Urological Institute, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.

    • William B Isaacs,
    • Alan K Meeker,
    • George Netto,
    • Angelo M De Marzo &
    • William G Nelson
  5. Center for Cancer Genomics, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.

    • Wennuan Liu &
    • Jianfeng Xu

Contributions

M.C.H. executed and analyzed all experiments and assisted in writing the manuscript. M.J.A. analyzed microarray data and assisted with statistical analysis of data. A.T., R.A., B.G., A.K.M., G.N. and A.M.D.M. assisted with execution and analysis of FISH, immunostaining and pathology experiments. D.M.E. assisted with execution of experiments. W.B.I., G.S.B., W.L. and J.X. contributed to analysis of microarray data in determination of prostate cancer TMPRSS2-ERG genomic breakpoints. W.G.N. assisted in experimental design and analysis and contributed to writing the manuscript. S.Y. conceived the study together with W.G.N., assisted in experimental design, execution and analysis and wrote the manuscript. All authors assisted in editing the manuscript.

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The authors declare no competing financial interests.

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