Prostate cancer is the second most common cause of male cancer deaths in the United States. Here we present the complete sequence of seven primary prostate cancers and their paired normal counterparts. Several tumors contained complex chains of balanced rearrangements that occurred within or adjacent to known cancer genes. Rearrangement breakpoints were enriched near open chromatin, androgen receptor and ERG DNA binding sites in the setting of the ETS gene fusion TMPRSS2-ERG, but inversely correlated with these regions in tumors lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumors contained rearrangements that disrupted CADM2, and four harbored events disrupting either PTEN (unbalanced events), a prostate tumor suppressor, or MAGI2 (balanced events), a PTEN interacting protein not previously implicated in prostate tumorigenesis. Thus, genomic rearrangements may arise from transcriptional or chromatin aberrancies to engage prostate tumorigenic mechanisms.

While recurrent gene fusions involving ETS family transcription factors are common in prostate cancer, their products are considered “undruggable” by conventional approaches. Recently, rare “targetable” gene fusions (involving the ALK kinase), have been identified in 1–5% of lung cancers1, suggesting that similar rare gene fusions may occur in other common epithelial cancers including prostate cancer. Here we employed paired-end transcriptome sequencing to screen ETS rearrangement negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF and ESRP1-RAF1 gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and MEK inhibitors. Screening a large cohort of patients, we found that although rare (1–2%), recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers, and melanoma. Taken together, our results emphasize the importance of RAF rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion harboring solid tumors, and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.

Chromosomal rearrangements account for all erythroblast transformation specific (ETS) family member gene fusions that have been reported in prostate cancer and have clinical, diagnostic and prognostic implications. Androgen-regulated genes account for the majority of the 5’ genomic regulatory promoter elements fused with ETS genes. TMPRSS2-ERG, TMPRSS2-ETV1 and SLC45A3-ERG rearrangements account for roughly 90% of ETS fusion prostate cancer. ELK4, another ETS family member, is androgen-regulated, involved in promoting cell growth, and highly expressed in a subset of prostate cancer, yet the mechanism of ELK4 over-expression is unknown. In this study, we identified a novel ETS family fusion transcript, SLC45A3-ELK4, and found it to be expressed in both benign prostate tissue and prostate cancer. We found high levels of SLC45A3-ELK4 mRNA restricted to a subset of prostate cancer samples. SLC45A3-ELK4 transcript can be detected at high levels in urine samples from men at risk for prostate cancer. Characterization of the fusion mRNA revealed a major variant in which SLC45A3 exon 1 is fused to ELK4 exon 2. Based on quantitative PCR analyses of DNA, unlike other ETS fusions described in prostate cancer, the expression of SLC45A3-ELK4 mRNA is not exclusive to cases harbouring a chromosomal rearrangement. Treatment of LNCaP cancer cells with a synthetic androgen (R1881) revealed that SLC45A3-ELK4, and not endogenous ELK4, mRNA expression is androgen-regulated. Altogether, our findings show that SLC45A3-ELK4 mRNA expression is heterogeneous, highly induced in a subset of prostate cancers, androgen-regulated, and most commonly occurs through a mechanism other than chromosomal rearrangement (e.g., trans-splicing).

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date, little is known about the presence of non-ETS fusion events in prostate cancer. We used next-generation transcriptome sequencing (RNA-seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and four involving non-ETS genes such as CDKN1A (p21), CD9, and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency, but, interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement-prone phenotype.

We have developed FusionSeq to identify fusion transcripts from paired-end RNA-sequencing. FusionSeq includes filters to remove spurious candidate fusions with artifacts, such as misalignment or random pairing of transcript fragments, and it ranks candidates according to several statistics. It also has a module to identify exact sequences at breakpoint junctions. FusionSeq detected known and novel fusions in a specially sequenced calibration data set, including eight cancers with and without known rearrangements.

Emerging molecular and clinical data suggest that ETS fusion prostate cancer represents a distinct molecular subclass, driven most commonly by a hormonally regulated promoter and characterized by an aggressive natural history. The study of the genomic landscape of prostate cancer in the light of ETS fusion events is required to understand the foundation of this molecularly and clinically distinct subtype. We performed genome-wide profiling of 49 primary prostate cancers and identified 20 recurrent chromosomal copy number aberrations, mainly occurring as genomic losses. Co-occurring events included losses at 19q13.32 and 1p22.1. We discovered 3 genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in non- rearranged prostate cancer is accompanied by gene expression deregulation in an independent dataset and by protein deregulation of MYO6. To analyze copy number alterations within the ETS genes, we performed a comprehensive analysis of all 27 ETS genes and of the 3Mbp genomic area between ERG and TMPRSS2 (21q) with an unprecedented resolution (30 bp). We demonstrate that high-resolution tiling arrays can be used to pin-point breakpoints leading to fusion events. This study provides further support to defining a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.

A step toward the molecular classification of prostate cancer was the discovery of recurrent erythroblast transformation-specific rearrangements, most commonly fusing the androgen-regulated TMPRSS2 promoter to ERG. The TMPRSS2-ERG fusion is observed in around 90% of tumors that overexpress the oncogene ERG. The goal of the current study was to complete the characterization of these ERG-overexpressing prostate cancers. Using fluorescence in situ hybridization and reverse transcription-polymerase chain reaction assays, we screened 101 prostate cancers, identifying 34 cases (34%) with the TMPRSS2-ERG fusion. Seven cases demonstrated ERG rearrangement by fluorescence in situ hybridization without the presence of TMPRSS2-ERG fusion messenger RNA transcripts. Screening for known 5' partners, we determined that three cases harbored the SLC45A3-ERG fusion. To discover novel 5' partners in these ERG-overexpressing and ERG-rearranged cases, we used paired-end RNA sequencing. We first confirmed the utility of this approach by identifying the TMPRSS2-ERG fusion in a known positive prostate cancer case and then discovered a novel fusion involving the androgen-inducible tumor suppressor, NDRG1 (N-myc downstream regulated gene 1), and ERG in two cases. Unlike TMPRSS2-ERG and SCL45A3-ERG fusions, the NDRG1-ERG fusion is predicted to encode a chimeric protein. Like TMPRSS2, SCL45A3 and NDRG1 are inducible not only by androgen but also by estrogen. This study demonstrates that most ERG-overexpressing prostate cancers harbor hormonally regulated TMPRSS2-ERG, SLC45A3-ERG, or NDRG1-ERG fusions. Broader implications of this study support the use of RNA sequencing to discover novel cancer translocations.

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5′ fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements.