Decision letter: SPOP mutation leads to genomic instability in prostate cancer

Abstract

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Genomic instability is a fundamental feature of human cancer often resulting from impaired genome maintenance. In prostate cancer, structural genomic rearrangements are a common mechanism driving tumorigenesis. However, somatic alterations predisposing to chromosomal rearrangements in prostate cancer remain largely undefined. Here, we show that SPOP, the most commonly mutated gene in primary prostate cancer modulates DNA double strand break (DSB) repair, and that SPOP mutation is associated with genomic instability. In vivo, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation resulting in impaired homology-directed repair (HDR) of DSB. Furthermore, we found that SPOP mutation sensitizes to DNA damaging therapeutic agents such as PARP inhibitors. These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that mutant SPOP may increase response to DNA-damaging therapeutics. https://doi.org/10.7554/eLife.09207.001 eLife digest Prostate cancer is the most common type of cancer in men in the UK and USA. Cancers develop when cells in the body acquire genetic mutations that allow the cells to grow rapidly and form a mass known as a tumor. Prostate cancer cells from different individuals can carry different genetic mutations, which affects whether the disease progresses and how the tumors respond to medical treatments. This genetic variety arises in cancer cells partly from a phenomenon known as genomic instability, in which DNA mutations accumulate due to defects in DNA repair. Genetic studies of biopsies taken from human prostate cancers have shown that genomic instability causes chromosomes—the structures in which the cell's DNA is organized—to break and then be stuck back together haphazardly. As a result, fragments of chromosomes can end up in the wrong position, be duplicated, or be lost altogether. All of these mutations could spur on the growth of the tumor. However, it is currently not clear why some prostate cancers are more genomically unstable than others, or what exactly causes this instability. Boysen, Barbieri et al. studied prostate cancer cells taken from patients before they started medical treatment. The experiments show that the cancer cells with high levels of genomic instability also often had mutations in a gene that encodes a protein called SPOP. These mutations occur in about 10 percent of men with prostate cancer and appear early in the development of the tumors. Next, they studied the SPOP protein in zebrafish (which is nearly identical to human SPOP), as well as in mouse and human cells. The experiments show that SPOP normally helps the cell to accurately repair DNA that has been damaged. Mutations in SPOP change the DNA repair process, which lead to genomic instability by increasing the likelihood that broken chromosomes will be stuck back together incorrectly. Further experiments tested drugs known as PARP inhibitors on mouse and human prostate cancer cells. The drugs, which have been recently tested successfully in patients with prostate cancer, block a different method of DNA repair that operates separately to the one that involves SPOP. When both of these pathways were inactivated—one by the SPOP mutation, the other by the drug—the cancer cells died more quickly. Therefore, men that are diagnosed with types of prostate cancer in which the gene that encodes SPOP is mutated might benefit from treatment with PARP inhibitors or other therapies that affect DNA repair. https://doi.org/10.7554/eLife.09207.002 Introduction Genomic instability is a fundamental feature of human cancer, and DNA repair defects resulting in impaired genome maintenance promote pathogenesis of many human cancers (Hanahan and Weinberg, 2011; Garraway and Lander, 2013). In prostate cancer, structural genomic rearrangements, including translocations (e.g., TMPRSS2-ERG) and copy number aberrations (e.g., 8q gain, 10q23/PTEN loss) are a key mechanism driving tumorigenesis (Visakorpi et al., 1995; Cher et al., 1996; Tomlins et al., 2005; Zhao et al., 2005; Liu et al., 2006; Demichelis et al., 2009; Beroukhim et al., 2010). Whole genome sequencing (WGS) has allowed an unprecedented insight into the alterations underlying cancer. Recently, WGS of treatment naive, clinically localized prostate cancer revealed a striking abundance of genomic rearrangements, in some samples comparable to the number of rearrangements in metastatic prostate cancer (Baca et al., 2013). Furthermore, the type (intrachromosomal vs interchromosomal) and complexity of rearrangements in these tumors shows remarkable heterogeneity, potentially suggesting distinct mechanisms of instability in different molecular classes of prostate cancer. However, somatic alterations underlying these phenomena remain unexplained. Mutations in SPOP (Speckle-type POZ protein) occur in around 10% of prostate cancers and represent the most common non-synonymous mutations in primary prostate cancer (Barbieri et al., 2012). SPOP mutations define a distinct molecular class of prostate cancer; they are mutually exclusive with ETS rearrangements but display distinct patterns of somatic copy number alterations (SCNAs) (Barbieri et al., 2012). Here, we investigated somatic alterations associated with genomic rearrangements in prostate cancer. We show that SPOP mutation is an early event specifically associated with increased intrachromosomal genomic rearrangements. Mechanistically, in vitro and in vivo data suggest that SPOP participates in repair of DNA double strand breaks (DSB), and SPOP mutation impairs homology-directed repair (HDR), instead promoting error-prone non-homologous end joining (NHEJ). Results To nominate somatic events associated with structural genomic rearrangements in clinically localized prostate cancer, we examined WGS data from 55 treatment naive prostate cancers (Baca et al., 2013) (Figure 1A). This analysis revealed a bimodal distribution, with a more common, ‘low-rearrangement’ population, and a less frequent ‘high-rearrangement’ population primarily driven by intrachromosomal rearrangements (deletions, inversions, and tandem duplications), rather than balanced interchromosomal rearrangements (Figure 1B). We then analyzed the association between recurrent somatic alterations (point mutations and SCNAs) and number of rearrangements (Figure 1C; Figure 1—figure supplements 1, 2). Several recurrent deletions, primarily on chromosomes 5q and 6q, were significantly associated with intrachromosomal genomic rearrangements (Figure 1—figure supplement 1), and these were completely distinct compared to alterations associated with interchromosomal rearrangements (Figure 1—figure supplement 2). Among recurrent point mutations, only a single lesion—mutation in SPOP—was significantly associated with increased rearrangements (Figure 1C). Consistent with increased intrachromosomal rearrangements, SCNA analysis revealed that SPOP mutant prostate cancers showed significantly higher total copy number alteration burden (Figure 1D). Figure 1 with 5 supplements see all Download asset Open asset SPOP mutant prostate cancer displays increased genomic rearrangements. (A) Distribution analysis of genomic rearrangements from 55 clinically localized prostate cancers distinguishes two subpopulations. (B) Increased total rearrangements are driven by intrachromosomal rather than interchromosomal rearrangements. 55 clinically localized prostate cancers ordered (right to left) according to total rearrangements; numbers of intrachromosomal and interchromosomal rearrangements are displayed. (C) Association of recurrent point mutations with intrachromosomal rearrangements. X-axis shows (−log10) p-value. (D) SPOP mutant prostate cancers harbor increased total somatic copy number aberration (SCNA) burden. The fraction of altered genome, partitioned into bins covering a range from <0.01 to ≥0.5, is shown as a histogram for SPOP WT and SPOP mutant tumors. Inset: the percentage of altered genome is significantly increased in SPOP mutant prostate cancers (p = 0.0016, two-sample Wilcoxon-Mann-Whitney test). (E) Frequency of somatic copy number alterations in 430 clinically localized prostate cancers. SPOP-mutant cancers (orange) and SPOP-wild-type tumors (gray). Length of bars indicates the frequency of copy number alterations. (F) Clonality of selected alterations associated with genomic rearrangements, in 430 clinically localized prostate cancers. https://doi.org/10.7554/eLife.09207.003 SPOP mutation frequently co-occurs with specific SCNAs, designating a molecular class of prostate cancer (Barbieri et al., 2012; Blattner et al., 2014) (Figure 1—figure supplement 3). Independent analysis of SCNAs from three publicly available data sets comprising 430 tumors (Baca et al., 2013; Barbieri et al., 2012; Consortium TCGA, 2015), including 47 SPOP mutant prostate cancers, confirmed that the rearrangement-associated deletions (Figure 1—figure supplement 1) were those enriched in SPOP mutant prostate cancer (Figure 1E). When individually comparing SPOP mutations and associated deletions (CHD1 and MAP3K7), we did not observe significant differences in SCNA burden for any one lesion (Figure 1—figure supplement 4). Analysis of clonality (Baca et al., 2013; Prandi et al., 2014) of specific lesions showed that SPOP mutations were highly clonal compared to loci in the associated deletion peaks, supporting that SPOP mutations precede deletions (Figure 1F). In addition, analysis of dependencies of the lesions supports SPOP mutations preceding CHD1 deletions; no lesions were predicted to precede SPOP mutation (Figure 1—figure supplement 5). Together, these data nominate a distinct prostate cancer class characterized by early SPOP mutations and genomic instability. We posited that the SPOP mutation impacts genome maintenance and prioritized SPOP for functional studies. We explored the functional role of SPOP in vivo, using zebrafish as a rapidly assessable vertebrate model system. SPOP is highly conserved (97.3% identical at the amino acid level between human and zebrafish, Figure 2—figure supplement 1A). Knockdown of Spop by two different splice-blocking morpholinos (MO5, MO7) dramatically impaired brain and eye development as well as decreased overall body size (Figure 2A,B; Figure 2—figure supplement 1F), resulted in gene expression changes consistent with p53 activation, and apoptosis measured by TUNEL assay (Figure 2C, Figure 2—figure supplement 1E). Microinjection of human SPOP mRNA rescued these phenotypes, confirming specificity of the morpholino effects (Figure 2A–C, Figure 2—figure supplement 1F). To nominate signaling pathways impacted by Spop, we performed transcriptional profiling using RNA-seq on zebrafish with Spop knockdown and ectopic expression of wild-type SPOP (SPOP-wt) and SPOP-F133V, the most commonly mutated residue in prostate cancer (Figure 2D). Consistent with recently reported proteomic data (Theurillat et al., 2014) and heterodimerization between mutant and wild-type SPOP in our models (Figure 2—figure supplement 2), transcriptional responses to SPOP-F133V compared with SPOP-wt and Spop morpholino showed a pattern consistent with dominant negative, selective loss of function; SPOP-F133V correlated with SPOP-wt for some gene sets (Cluster B) and correlated with Spop morpholino for others (Cluster A) (Figure 2D, Figure 2—source data 1). Gene set enrichment analyses (GSEA) revealed gene sets involving DNA repair impacted by modulating SPOP function (Figure 2—source data 2). Notably, the transcriptional response to F133V correlated highly with BRCA1 inactivation (Figure 2E). While SPOP has been previously proposed as involved in DNA damage and repair (DDR) signaling based on in vitro experiments (Zhang et al., 2014a), these results for the first time implicate a functional role for SPOP in DDR signaling in vivo and suggest this function is selectively impaired by prostate cancer-derived SPOP mutations. To test this hypothesis in human prostate cancers, we performed unsupervised hierarchical clustering of transcriptional data from 11 SPOP mutant and 53 SPOP wild-type tumors, based on the transcriptional signature of BRCA1 inactivation (MSigDB: M2748). We observed significant segregation of SPOP mutant tumors (p-value = 9.5e−07, Figure 2F) using this signature, with less robust segregation of tumors harboring CHD1 or MAP3K7 deletions co-occurring with SPOP mutation (Figure 2—figure supplement 3). These data suggest that human prostate cancers with SPOP mutations show transcriptional effects similar to BRCA1 inactivation, consistent with a role for SPOP in DSB repair. Supporting this hypothesis, we observed a trend of mutual exclusivity between SPOP mutations and BRCA1 alterations in genomic data (p-value = 0.0496, Figure 2—figure supplement 4). Figure 2 with 4 supplements see all Download asset Open asset SPOP mediates DNA damage repair. (A) Evaluation of SPOP function during zebrafish development. Phenotype of morpholino-mediated Spop knockdown (MO5) in zebrafish embryos at 70 hr post fertilization (hpf). Injection of human SPOP mRNA (250 pg) rescued the phenotype. (B) Quantification of the rescue of SPOP phenotype after ectopic expression of human SPOP mRNA. Results are represented as s.e.m. (C) Whole mount TUNEL assay to determine apoptosis in zebrafish embryos. Arrows point to apoptotic cells (brown). Shown are representative images. (D) Heatmap representation of gene expression differences in zebrafish embryos ectopically expressing SPOP-wt or SPOP-F133V compared to SPOP knockdown by morpholino (MO). The list of genes can be found in Figure 2—figure supplement 3. Number of genes per block: A (198), B (429), C (223). (E) Gene set enrichment analysis (GSEA) of RNA sequencing data derived from zebrafish embryos expressing SPOP-wt or SPOP-F133V (24 hpf). Enrichment plot for the BRCA1 gene signature is shown. Molecular Signatures Database (MSigDB) systematic name indicated in brackets. (NES) Normalized Enrichment Score. (FDR) False Discovery Rate. (F) Dendrogram of human primary prostate cancer cases based on BRCA1 knockdown genes (MSigDB: M2748). Unsupervised clustering of RNA-seq data from human primary prostate cancer with wild-type (n = 53) or mutant SPOP (n = 11), performed on the BRCA1 knockdown gene signature (M2748) identified in zebrafish embryos by GSEA as shown in (E). https://doi.org/10.7554/eLife.09207.009 Figure 2—source data 1 List of genes contained in blocks A, B, and C in the heatmap in Figure 2D. Provided as excel file. https://doi.org/10.7554/eLife.09207.010 Download elife-09207-fig2-data1-v2.xls Figure 2—source data 2 Results from GSEA comparing zebrafish embryos ectopically expressing SPOP-wt or -F133V. Provided as excel file. https://doi.org/10.7554/eLife.09207.011 Download elife-09207-fig2-data2-v2.xlsx Consistent with the hypothesis that SPOP is involved in DSB repair in prostate cells, SPOP forms nuclear foci in prostate cells after γ-irradiation (Figure 3A) and stable expression of SPOP-wt conferred resistance to DSB-inducing agents cisplatin and camptothecin (CPT) (Figure 3—figure supplement 1A,B). To further define the impact of SPOP mutation in response to DNA damage, we utilized primary prostate cells isolated from transgenic mice with Cre-dependent conditional expression of SPOP-F133V. After transduction with Tamoxifen-inducible Cre (Cre-ERT2), cells were treated with 4-OH tamoxifen or vehicle and exposed to ionizing radiation (IR) (Figure 3—figure supplement 2). Spop localized in nuclear foci similar to human LNCaP cells in mouse prostate cells (MPCs) after IR, with no alteration in foci by expression of SPOP-F133V (Figure 3A, Figure 3—figure supplement 1I). Induction of SPOP-F133V resulted in delayed recovery from IR-induced damage as measured by comet assay (Figure 3B). No differences were seen in apoptosis after IR, as measured by PARP cleavage (Figure 3—figure supplement 2), subG1 events, or caspase-3 cleavage (data not shown). To functionally characterize how SPOP mutations affect response to DSB, we expressed SPOP-wt and SPOP-F133V in benign prostate epithelial cells (RWPE) and prostate cancer cells (22Rv1), and examined the induction, recognition, and resolution of CPT-induced DSBs. We found that DSB formation (measured by γH2AX foci and protein levels) was not affected by modulating SPOP function with siRNA, or expression of wildtype or mutant SPOP (Figure 3C, Figure 3—figure supplement 1C,D). Furthermore, there were no observed differences in early DNA damage signaling events, such as phosphorylation of ATM, ATR, Chk1, and Chk2 (Figure 3—figure supplement 1E–H), indicating that SPOP did not affect initial induction and recognition of DSBs or initial steps in DDR signaling. Consistent with this, SPOP showed only limited co-localization with early markers of DSB (γH2AX, phospho-ATM) in irradiated prostate cells (Figure 3—figure supplement 1I). Figure 3 with 3 supplements see all Download asset Open asset SPOP mutation impairs HDR and promotes NHEJ and SPOP-wt modulates DSB repair activity similar to BRCA1. (A) SPOP forms nuclear foci after induction of DNA damage by γ-irradiation (3GY) in prostate cells derived from transgenic mice (MPC) and human LNCaP cells. Red represents nuclear Spop protein foci. Blue represents nuclear DNA stained with DAPI. (B) MPC expressing Cre-inducible SPOP-F133V was infected with tamoxifen-inducible Cre (CreERT2), and DNA damage was assessed after IR with comet assays. Inset: representative cells showing comet tails after IR. (C, D, E) Quantification of γH2AX, 53BP1, or RAD51 foci in RWPE cells overexpressing WT or F133V mutant SPOP after camptothecin (CPT) (1 μM) induced DNA damage. Time indicates the observation interval in minutes including double strand break (DSB) induction (0–60 min) and recovery (60–180 min). Shown are the percentages of cells for each genotype with more than 5 foci per nucleus. Results are represented as s.e.m. (F) Representative pictures showing γH2AX, RAD51, or 53BP1 foci (red or green). Blue represents nuclear DNA stained with DAPI. (G) Quantification of Rad51 foci in γ-irradiated (2GY) MPC with tamoxifen-inducible SPOP-F133V. Rad51 foci were counted 30 min post irradiation. (H) Representative pictures showing Rad51 foci in mouse prostate epithelial cells before and after γ-irradiation (2GY). (I) Quantification of RAD51 foci in RWPE cells treated with siSPOP or control siRNA and subsequently exposed to CPT (1 μM, 1 hr). https://doi.org/10.7554/eLife.09207.016 We next examined specific markers (53BP1, RAD51) of the two major DSB repair pathways, HDR and NHEJ. RAD51 is a component of the HDR pathway and a marker for engagement of the HDR machinery (Baumann et al., 1996). 53BP1 is a positive regulator of NHEJ that blocks 5′-DNA-end resection and therefore functions at the intersection of HDR and NHEJ; if 53BP1 is not cleared from sites of DSB by HDR components, it promotes error prone NHEJ (Panier and Boulton, 2014). Strikingly, SPOP-F133V-expressing prostate cells showed delayed clearance of 53BP1 from sites of DSB (Figure 3D,F, Figure 3—figure supplement 3A); similar effects were seen with another SPOP mutation (F102C) commonly observed in prostate cancer (Figure 3—figure supplement 3I,J). Furthermore, SPOP-wt increased RAD51 foci formation compared to controls, while SPOP-F133V-expressing cells showed a dramatic decrease in RAD51 foci formation (Figure 3E,F, Figure 3—figure supplement 3B), consistent with impairment of HDR. Induction of SPOP-F133V in primary MPCs similarly decreased Rad51 foci after IR (Figure 3G,H). Knockdown of SPOP also resulted in a decrease of RAD51 foci, suggesting a selective loss of function of SPOP-F133V in HDR (Figure 3I). We also observed decreased clearance of 53BP1 foci and decreased RAD51 foci formation in SPOP-F133V-expressing cells after gamma irradiation, indicating that this effect is not specific to one mechanism of DSB induction (Figure 3—figure supplement 3C–H). The observed changes in DSB repair were not accompanied by changes in the cell cycle distribution of cells expressing SPOP-wt or F133V under these conditions (Figure 3—figure supplement 3K). We next investigated the role of SPOP in DSB repair using the well established DR-GFP and Pem1-Ad2-EGFP reporter assays as functional readouts for HDR and NHEJ, respectively (Figure 4A,D) (Pierce et al., 1999; Seluanov et al., 2004). In the DR-GFP assay, knockdown of SPOP by siRNA decreased HDR competence in human epithelial cells to a similar level of BRCA1 knockdown (Figure 4B). Conversely, ectopically expressed SPOP-wt increased the HDR competence in these cells, with partial loss of this function by mutant SPOP (Figure 4C). In contrast, the Pem1-Ad2-EGFP NHEJ reporter assay indicated an increase of NHEJ activity in SPOP-F133V expressing epithelial cells, while both SPOP siRNA and BRCA1 siRNA increased NHEJ (Figure 4E,F). Taken together, these results suggest that SPOP promotes HDR, while somatic mutation in SPOP, as observed in prostate cancer with increased genomic rearrangements, impairs HDR and promotes error-prone NHEJ. Figure 4 Download asset Open asset SPOP modulates DSB repair activity similar to BRCA1. (A) Schematic overview of the DR-GFP assay used to measure homology-directed repair (HDR) activity. (B, C) Analysis of the HDR-activity in HEK 293 cells with siRNA knockdown of SPOP or BRCA1 and ectopically expressing SPOP-wt or SPOP-F133V. (D) Schematic overview of the Pem1-Ad2-EGFP assay used to measure NHEJ-activity. (E, F) Analysis of the NHEJ-activity in HEK 293 cells with siRNA knockdown of SPOP or BRCA1 and ectopically expressing SPOP-wt or SPOP-F133V. All results are represented as s.e.m. https://doi.org/10.7554/eLife.09207.020 Human cancers with underlying defects in HDR (such as BRCA1 inactivated breast and ovarian cancers) (Bryant et al., 2005; Farmer et al., 2005; Audeh et al., 2010; Tutt et al., 2010) show sensitivity to poly (ADP-ribose) polymerase 1 (PARP) inhibition. To test if SPOP inactivation conferred sensitivity to PARP inhibition after DSB induction, we utilized siRNA targeting SPOP in human prostate cancer cell lines (PC-3, LnCap, 22Rv1), followed by irradiation (5GY) and incubation with the PARP inhibitors olaparib or veliparib. Reduction of SPOP expression increased the sensitivity of these prostate cancer cells to both PARP inhibitors (Figure 5A–C, Figure 5—figure supplement 1B,C). To test if SPOP mutation also sensitizes to PARP inhibition, we treated MPCs (control vs tamoxifen-induced SPOP-F133V) with olaparib followed by IR—expression of SPOP-F133V increased sensitivity to olaparib similar to loss of SPOP in human prostate cancer cell lines (Figure 5D). This was further confirmed in 22Rv1 cells ectopically expressing SPOP-F133V, which also showed an increased sensitivity to olaparib in viability assays, while cells ectopically expressing SPOP-wt were relatively resistant (Figure 5—figure supplement 1A). We further confirmed these results in HEK293 cells stably overexpressing the two common SPOP mutants (Y87N, F133V) in clonogenic survival assays (Figure 5E,F). Figure 5 with 1 supplement see all Download asset Open asset SPOP mutation sensitizes cells to therapeutic PARP inhibition. (A–D) Analysis of sensitivity to the PARP inhibitor olaparib in irradiated (5GY) prostate cancer (PC-3, LNCaP, 22Rv1) and mouse prostate epithelial cells (MPC) after SPOP knockdown (siSPOP) or tamoxifen-inducible SPOPF133V expression. BRCA1 knockdown (siBRCA1) and non-targeting siRNA (sictrl) served as positive or negative control. The IC50 of olaparib for each genotype is indicated in Molar (M). (E) Analysis of the sensitivity of most frequently occurring prostate-specific SPOP mutants Y87N and F133V to olaparib in clonogenic assays using HEK293 cells. (F) Representative examples from the clonogenic assay used to assess long-term survival in HEK293 cells stably expressing SPOP-wt or SPOP mutants. (G) Impact of SPOP mutation on induction of genomic instability in 22Rv1 cells after olaparib (1 μM) treatment as measured by comet assay. Increased genomic instability was measured by an increase in the tail moment. (H) Genomic instability in tamoxifen-inducible SPOP-F133V expressing mouse prostate cells after γ-irradiation (15GY) with and without olaparib (1 μM) treatment as measured by comet assay. All results are represented as s.e.m. https://doi.org/10.7554/eLife.09207.021 To determine if the altered sensitivity to PARP inhibitors was associated with increased DNA damage, consistent with impaired double-strand break repair, we performed single-cell gel electrophoresis (comet assay) in prostate cells treated with olaparib. The comet assay revealed an increase of genomic instability in SPOP-F133V RWPE cells after PARP inhibition, similar to SPOP (and BRCA1) knockdown with siRNA (Figure 5G). Similarly, primary MPCs expressing SPOP-F133V showed increased damage after olaparib treatment (Figure 5H). Taken together, these data argue that SPOP mutation confers sensitivity to PARP inhibition due to impaired error-free HDR DSB repair and an increase in error-prone NHEJ. Discussion In summary, here we report that SPOP, the substrate recognition component of an E3 ubiquitin ligase, is a regulator of the HDR-based DSB repair machinery. Using functional genetic approaches, we find that prostate-specific SPOP mutants deregulate DSB repair by promoting the error-prone NHEJ pathway (Figure 6). Importantly, after DSB induction, this loss of function leads to an increased sensitivity of mutant SPOP prostate cancer cells to PARP inhibition. These observations provide the first mechanism for the increased genomic instability of SPOP mutant prostate cancer, but also suggest that similar to other cancers with impaired HDR, this distinct class of cancer might benefit from treatment with clinically established DNA damaging therapeutics. This study therefore provides a rationale for hypothesis-based biomarker-driven clinical trials using PARP inhibitors or other DNA damaging agents in patients with prostate cancer. Figure 6 Download asset Open asset Proposed model of the effects of SPOP mutation on genome instability. In prostate epithelial cells, SPOP-wt promotes error-free HR and maintains genome stability. SPOP mutation impairs HR repair and promotes error-prone NHEJ, leading to increased genomic instability. https://doi.org/10.7554/eLife.09207.023 Genomic rearrangements represent critical events deregulating prostate cancer genomes and driving tumorigenesis, although among individual cancer samples, there is marked variability in number and character of structural rearrangements. Here, we identified a genomically unstable subclass of primary prostate cancer characterized by dramatically increased intrachromosomal rearrangements. This subset of clinically localized, treatment-naive prostate cancers display a degree of genomic instability previously thought only to occur in late stage, metastatic tumors. Importantly, increased rearrangements likely result in higher total SCNA burden, which is associated with more aggressive clinical behavior (Hieronymus et al., 2014; Lalonde et al., 2014). Increased rearrangements were associated with mutations in SPOP, encoding the substrate-recognition component of an E3 ubiquitin ligase complex. SPOP mutations are the most common point mutations in prostate cancer, but their role in promoting prostate cancer pathogenesis remains unclear. SPOP mutations occur early in the history of prostate cancer, based on clonality analysis reported here and presence in the prostate cancer precursor high grade prostatic intraepithelial neoplasia (HG-PIN) (Barbieri et al., 2012), potentially consistent with a ‘gatekeeper’ role in genome maintenance. Consistent with increased intrachromosomal rearrangements in SPOP mutant cancers, in vivo data nominated a functional role for SPOP in DSB repair, similar to BRCA1. Interestingly, SPOP loss of function induced a developmental phenotype of neuronal degeneration and apoptosis; similar effects have been reported with other genes modulating DNA repair, including BRCA1 (Pulvers and Huttner, 2009). SPOP encodes the substrate recognition component of an E3 ubiquitin ligase, and here we identified a role for SPOP in regulating the HDR-based DSB repair machinery. Notably, many established components of the DNA damage response are components of enzymes regulating ubiquitylation (Ceol et al., 2011). A number of substrates have been reported as deregulated by prostate cancer-derived SPOP mutations, but the relevance of these in vitro findings to human prostate cancers is still unclear (Geng et al., 2013, 2014; An et al., 2014; Theurillat et al., 2014; Zhang et al., 2014b). In contrast, we focused on the phenotypes observed in human prostate cancers harboring SPOP mutations. Using functional genetic approaches, we find that prostate-specific SPOP mutants deregulate DSB repair by promoting the error-prone NHEJ pathway (Figure 4F).