Figure 5.

Androgen-induced TOP2B mediated DSB 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 evidenced 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, While both showed similar recombination frequency at the vector backbone, recombination frequency within the IN-1 insert was significantly higher than that in the EX-6 insert in pooled colonies as determined using the strategy shown on the right. Data are shown as mean ± SE 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-PK_CS (Wort, si-DNA-PK_CS), 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.