Nuclear receptor-induced chromosomal proximity and DNA breaks underlie specific translocations in cancer

Abstract

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.

Figure 1. Liganded-AR and Genotoxic Stress Synergistically Induce Chromosomal Translocations in Prostate Cancer Cells

(A and B) Identification and characterization of induced TMPRSS2:ERGb and TMPRSS2:ETV1b translocations in LNCaP cells. Top: schematic structures for the TMPRSS2, ERG, and ETV1 mRNA indicating exon positions. Middle: RT-PCR amplification of TMPRSS2:ERGb (A) or TMPRSS2:ETV1b (B) fusion transcripts from 48 individual cell samples. Bottom: confirmation of position and fusion sites by automated DNA sequencing. (C and D) Statistical analysis of DHT and IR induced TMPRSS2:ERG (C) and TMPRSS2:ETV1 (D) translocations (n=3, ±SEM). (E and F) Identification and characterization of induced TMPRSS2:ERG (E) and TMPRSS2:ETV1 (F) translocation isoforms in LNCaP cells. Bottom: summary of distinct fusion types. (G and H) Involvement of DNA repair machinery in induced TMPRSS2:ERGb and TMPRSS2:ETV1b translocations by QPCR with indicated siRNAs (n=3, ±SEM).

Figure 2. AR-Induced and Motor-Dependent Chromosomal Interactions of TMPRSS2 and ERG or ETV1 Loci

(A and B) Interphase FISH analysis on PrEC cells with TMPRSS2 (green), ERG (red) (A), or ETV1 (red) (B) probes. (C and D) Actin polymerization-dependent interchromosomal interactions. (E and F) Nuclear myosin-dependent interchromosomal interactions. (G) ATPase activity of NMI is required for DHT induced interchromosomal interactions. (H and I) Requirement of nuclear myosin/actin motor system for induced TMPRSS2:ERGb and TMPRSS2:ETV1b translocations in LNCaP cells pretreated with Latrunculin (LtA) or Jasplakinolide (Jpk) (H) or transfected with NMI siRNA (I) (n=3, ±SEM).

Figure 3. Identification of Breakpoints for TMPRSS2:ERG and TMPRSS2:ETV1 Translocations

(A) The tracks display the human genome coordinates (hg18 assembly), red band: predicted potential DSBs based on ChIP-seq using anti-BrdU antibodies (see Experimental Procedures). Boxes: exons; lines: introns; Region I to IV: DNA break points within corresponding loci. (B) The fold change of the tag density in the double-strand break regions I, II, III and IV after DHT treatment. (C and D) Conventional ChIP analysis with anti-BrdU antibodies on ERG and TMPRSS2 intronic break regions identified by ChIP-seq. (E-G) Identification and characterization of induced TMPRSS2:ERG and TMPRSS2:ETV1 translocation breakpoints. Top: genomic DNA extracted from LNCaP cells either non-transfected (E) or co-transfected with MeCP2 siRNA and FLAG-DOT1L expression vector (F and G) was subjected to PCR amplification using primers flanking Region II and I (E), Region III and I (F), or Region IV and I (G). Bottom: automated DNA sequencing aligned to ERG or ETV1 (green) and TMPRSS2 (orange) with genomic position of starting and ending nucleotides shown. Red box: common sequence shared by TMPRSS2 and ERG or ETV1. (H and I) Graphic illustration of translocation patterns corresponding to induced TMPRSS2:ERG (H) and TMPRSS2:ETV1 (I) translocations. Potential break/fusion sites are shown as Red oval: TMPRSS2:ERG; blue oval: TMPRSS2:ETV1; dot line: distinct fusion patterns.

Figure 4. AR-Dependent Local Chromatin Structural Alteration Sensitizes to Site-Specific Genotoxic Stress-Induced DSBs

(A-E) Top: schematic diagram showing the relative positions of break/fusion sites and potential AREs located on ERG and TMPRSS2 loci. Blue boxes, potential AREs; red boxes, break/fusion sites; black arrows, relative positions of PCR primers. Bottom: LNCaP cells were treated with DHT (10⁻⁷ M) (A), DHT (10⁻⁷ M) (B), IR (50 Gy) (C) or both (D) and DHT (10⁻⁷ M) (E) for time courses as indicated. ChIP analyses were performed with indicated antibodies on indicated regions (n=2, ±SEM). (F) Examination of TMPRSS2:ERGb and TMPRSS2:ETV1b fusion transcripts with RPA2 siRNAs (n=3, ±SEM).

Figure 5. Mechanisms that Initiate Extended DNA Breaks in AR-Dependent Tumor Translocations

(A-C) Induction of AID expression by AR agonist and genotoxic stress. (n=3, ±SEM). (D) DHT-dependent interaction between AR and AID. Immunoprecipitates of anti-AR were subjected to immunoblotting analysis with indicated antibodies. Immunoblotting of E2F1 was included as negative control. (E) Ligand-dependent Myc-AID recruitment to AR-binding sites (n=2, ±SEM). (F and G) The recruitment of AID and Gadd45 to AR binding sites is mediated by liganded receptor. LNCaP cells were treated with ethanol (EtOH), DHT (10⁻⁷ M), or Bicalutamide (CDX, 10 μM) for 1hr followed by ChIP analyses with anti-Myc (F) or anti-AR and anti-Gadd45 (G) antibodies on indicated regions (n=2, ±SEM). (H and I) AID contributes to DSBs generation. ChIP analyses were performed on control siRNA or AID siRNA transfected, DHT+IR treated (4hr) LNCaP cells with anti-Ku80 antibodies (n=2, ±SEM) (H) or anti-BrdU antibody following BrdU labeling by TdT (I) on indicated regions. (J) Examination of TMPRSS2:ERGb and TMPRSS2:ETV1b fusion transcripts in LNCaP cells transfected with AID siRNA (n=3, ±SEM). (K) Left: representative agarose gels with PCR products corresponding to TMPRESS2:ERGa translocations (as illustrated in Figure 3H band a). The genomic DNA of control or AID siRNA transfected LNCaP cells were subjected to PCR using primers flanking ligation site (red oval). Right: Statistical analysis (n=3, ±SEM).

Figure 6. Protective Effects of PIWIs on Chromosomal Translocation

(A) Left: summary of identified RGYW/WRCY motif related mutation. The fusion chromatins of ERG region A in control siRNA and AID siRNA samples or ERG region B were amplified as in Figure 3E. Right: statistics analysis (n=3, ±SEM). (B) Ligand-dependent UNG recruitment to intronic regions of TMPRSS2 and ERG loci. (C) Recruitment of UNG to translocation regions is AID dependent. D) Quantitation of induced TMPRSS2:ERGb and TMPRSS2:ETV1b fusion transcripts in LNCaP or PrEC cells. NPT: normal prostate tissue. (E) Statistical comparison of induced TMPRSS2:ERGb (left) and TMPRSS2:ETV1b (right) fusion transcripts LNCaP and PrEC cells (48 samples per group and n=3, ±SEM). (F) The relative expression level of PIWIL1 in LNCaP and PrEC cells. (G) The expression level of LINE-1 ORF2 was examined in PrEC cells transfected with indicated siRNAs. (H) PIWIL1 knockdown enhances γH2AX enrichment at intronic ERG and TMPRSS2 break/fusion sites. (I and J) Examination of TMPRSS2:ERGb and TMPRSS2:ETV1b fusion transcripts in LNCaP cells electroporated with PIWIL1 siRNA (I) or indicated plasmids (J) (n=3, ±SEM). (K) Left: representative agarose gels with PCR products corresponding to TMPRESS2:ERGa translocations (as illustrated in Figure 3H band a). Right: Statistical analysis (n=3, ±SEM).

Figure 7. PIWI Regulated LINE-1 ORF2 Endonuclease Contributes to Chromosomal Translocations

(A) The relative expression level of LINE-1 ORF2 in LNCaP and PrEC cells. (B) IR-dependent induction of LINE-1 ORF2 expression (n=3, ±SEM). (C) Overexpression of LINE-1 ORF2 enhances Ku80 enrichment at intronic ERG and TMPRSS2 break/fusion sites. (D) Examination of TMPRSS2:ERGb and TMPRSS2:ETV1b fusion transcripts in LNCaP cells electroporated with indicated plasmids (n=3, ±SEM). (E and F) ORF2 contributes to DSBs generation independent of AID. ChIP analyses with anti-Ku80 (E) or anti-AID (F) antibodies were performed in LNCaP cells electroporated with indicated plasmids (n=2, ±SEM). (G) Recruitment of LINE-1 ORF2 to translocation regions. ChIP analyses with anti-FLAG followed by anti-ORF2 antibodies were performed in LNCaP cells electroporated with FLAG-ORF2 plasmid (n=2, ±SEM). (H) The recruitment of ORF2 is independent of AID. ChIP analyses with anti-FLAG antibodies were performed in LNCaP cells electroporated with AID siRNA and FLAG-ORF2 plasmid (n=2, ±SEM). (I) Schematic illustration of molecular mechanisms of nuclear receptor-dependent non-random chromosomal translocations.