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Induced Chromosomal Proximity and Gene Fusions in Prostate Cancer

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Science  27 Nov 2009:
Vol. 326, Issue 5957, pp. 1230
DOI: 10.1126/science.1178124

Abstract

Gene fusions play a critical role in cancer progression. The mechanisms underlying their genesis and cell type specificity are not well understood. About 50% of human prostate cancers display a gene fusion involving the 5′ untranslated region of TMPRSS2, an androgen-regulated gene, and the protein-coding sequences of ERG, which encodes an erythroblast transformation–specific (ETS) transcription factor. By studying human prostate cancer cells with fluorescence in situ hybridization, we show that androgen signaling induces proximity of the TMPRSS2 and ERG genomic loci, both located on chromosome 21q22.2. Subsequent exposure of the cells to gamma irradiation, which causes DNA double-strand breaks, facilitates the formation of the TMPRSS2-ERG gene fusion. These results may help explain why TMPRSS2-ERG fusions are restricted to the prostate, which is dependent on androgen signaling.

Gene fusions are a hallmark of cancer development (1), but the mechanisms underlying their genesis and cell type specificities are unclear (2).

Fusions of the TMPRSS2 and ERG genes, which are located 3 Mb apart on human chromosome 21q22.2, are found in about 50% of prostate cancers and consist of the 5′ untranslated region of TMPRSS2, an androgen-regulated gene, fused to the protein-coding sequences of ERG, which encodes an erythroblast transformation–specific (ETS) transcription factor (2). Recently, estrogen was shown to induce rapid chromosomal movements that bring together estrogen receptor α–bound genes on different chromosomes (3). Given the broad similarities between estrogen and androgen signaling, we hypothesized that androgen might likewise induce chromosomal movements and bring together the TMPRSS2 and ERG genes, thereby facilitating the formation of gene fusion.

To examine the effects of androgen signaling on the formation of TMPRSS2-ERG, we studied LNCaP prostate cancer cells, which are androgen sensitive but lack this fusion gene (4) (fig. S1). We treated these cells with the androgen receptor (AR) ligand dihydrotestosterone (DHT) for 60 min. With use of dual-color fluorescence in situ hybridization (FISH), we observed that stimulation with DHT induced proximity between TMPRSS2 and ERG genomic loci (34/300 and 62/300 nuclei for ethanol and DHT, respectively, P = 0.003, χ2 test) (Fig. 1, A and B, and fig. S2). This effect was dependent on AR (fig. S3). Importantly, androgen did not induce proximity between the TMPRSS2 and ERG loci in DU145 human prostate cancer cells, which are androgen insensitive (4/197 and 8/197 nuclei for ethanol and DHT, respectively, P = 0.38, χ2 test).

Fig. 1

(A) FISH-based evaluation of induced proximity between TMPRSS2 (green) and ERG (red) on stimulation with 100 nM DHT in LNCaP cells. Colocalization is indicated by yellow arrows. Scale bars indicate 2 μm. (B) Induced proximity between TMPRSS2 and ERG in DU145 and LNCaP cells is quantified and represented as the percentage of nuclei exhibiting colocalization signals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) SYBR Green (Molecular Probes, Eugene, Oregon) and TaqMan (Applied Biosystems, Foster City, California) quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the TMPRSS2-ERG fusion transcript. (D) Gel-based RT-PCR analysis with primers spanning the first exon of TMPRSS2 and the sixth exon of ERG for representative clones. (E) FISH analysis of the ERG locus. Split signals representing ERG rearrangement are highlighted by arrows.

To determine whether this induced proximity facilitates the formation of gene fusions, we treated LNCaP cells with DHT for 12 hours and then irradiated the cells to induce DNA double-strand breaks. After irradiation [1 or 3 grays (Gy)], single cells were seeded in multiple 96-well plates by using flow cytometry and were clonally expanded. TMPRSS2-ERG fusion transcripts were detected in 25% (3/12) of the clones treated with 3-Gy irradiation but in only 2.3% (1/43) of those treated with 1 Gy (Fig. 1, C and D, fig. S4, and table S1). The expression of TMPRSS2-ERG transcripts in positive LNCaP clones was similar to that in VCaP prostate cancer cells (3), which endogenously harbor the gene fusion. Furthermore, TMPRSS2-ERG–expressing LNCaP cells showed evidence consistent with chromosomal aberrations at the ERG locus (Fig. 1E and fig. S5). Parental LNCaP cells have four copies of chromosome 21, generating four yellow signals. LNCaP cells that harbor TMPRSS2-ERG transcripts generate three yellow signals and a pair of red and green signals, suggestive of a rearrangement at the ERG locus.

Our results build on earlier work documenting the role of chromosomal proximity in gene fusions (5) and, in so doing, provide a conceptual framework for the genesis of gene fusions in hormone-regulated epithelial cancers. Androgen-induced proximity between TMPRSS2 and ERG could help explain why TMPRSS2-ERG fusions are restricted to the prostate, which is uniquely dependent on androgen signaling. We speculate that androgen signaling colocalizes the 5′ and 3′ gene fusion partners, thereby increasing the probability of a gene fusion when subjected to agents that cause DNA double-strand breaks.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1178124/DC1

Materials and Methods

Figs. S1 to S4

Table S1

References and Notes

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This work was supported by NIH grants P50CA69568, R01CA132874, and DOD W81XWH-09-2-0014. A.M.C. is a paid consultant to Gen-Probe, Incorporated. A.M.C. and S.A.T. are named as co-inventors on a patent related to gene fusions in prostate cancer that is licensed to Gen-Probe, Incorporated.
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