The genomic complexity of primary human prostate cancer.

Prostate cancer is the second most common cause of male cancer deaths in the United States. However, the full range of prostate cancer genomic alterations is incompletely characterized. Here we present the complete sequence of seven primary human prostate cancers and their paired normal counterparts. Several tumours contained complex chains of balanced (that is, 'copy-neutral') 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 tumours lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumours contained rearrangements that disrupted CADM2, and four harboured events disrupting either PTEN (unbalanced events), a prostate tumour 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 and engage prostate tumorigenic mechanisms.

Functional characterization of BC039389-GATM and KLK4-KRSP1 chimeric read-through transcripts which are up-regulated in renal cell cancer.

BACKGROUND: Chimeric read-through RNAs are transcripts originating from two directly adjacent genes (<10 kb) on the same DNA strand. Although they are found in next-generation whole transcriptome sequencing (RNA-Seq) data on a regular basis, investigating them further has usually been refrained from. Therefore, their expression patterns or functions in general, and in oncogenesis in particular, are poorly understood. RESULTS: We used paired-end RNA-Seq and a specifically designed computational data analysis pipeline (FusionSeq) to nominate read-through events in a small discovery set of renal cell carcinomas (RCC) and confirmed them in a larger validation cohort. 324 read-through events were called overall; 22/27 (81%) selected nominees passed validation with conventional PCR and were sequenced at the junction region. We frequently identified various isoforms of a given read-through event. 2/22 read-throughs were up-regulated: BC039389-GATM was higher expressed in RCC compared to benign adjacent kidney; KLK4-KRSP1 was expressed in 46/169 (27%) RCCs, but rarely in normal tissue. KLK4-KRSP1 expression was associated with worse clinical outcome in the patient cohort. In cell lines, both read-throughs influenced molecular mechanisms (i.e. target gene expression or migration/invasion) in a way that counteracted the effect of the respective parent transcript GATM or KLK4. CONCLUSIONS: Our data suggests that the up-regulation of read-through RNA chimeras in tumors is not random but causes regulatory effects on cellular mechanisms and may impact patient survival.

Discovery of non-ETS gene fusions in human prostate cancer using next-generation RNA sequencing.

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.

Testing mutual exclusivity of ETS rearranged prostate cancer.

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.

Distinct genomic aberrations associated with ERG rearranged prostate cancer.

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 three genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in nonrearranged 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 3 Mbp 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 define a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.

Identification of molecular tumor markers in renal cell carcinomas with TFE3 protein expression by RNA sequencing.

TFE3 translocation renal cell carcinoma (tRCC) is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study, we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq) as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNA read-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2) point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs). The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P < .001) and 22 papillary RCCs (P < .05-.07). Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) and Contactin 3 (CNTN3) expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both).

Characterization of KRAS rearrangements in metastatic prostate cancer.

UNLABELLED: Using an integrative genomics approach called amplification breakpoint ranking and assembly analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, a specific knockdown of which attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 mitogen-activated protein kinase (MAPK) pathways. This is the first report of a gene fusion involving the Ras family, suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. SIGNIFICANCE: This is the first description of an oncogenic gene fusion of KRAS, one of the most studied proto-oncogenes. KRAS rearrangement may represent the driving mutation in a rare subset of metastatic prostate cancers, emphasizing the importance of RAS-RAF-MAPK signaling in this disease.

FusionSeq: a modular framework for finding gene fusions by analyzing paired-end RNA-sequencing data.

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.

Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma.

Although recurrent gene fusions involving erythroblastosis virus E26 transformation-specific (ETS) family transcription factors are common in prostate cancer, their products are considered 'undruggable' by conventional approaches. Recently, rare targetable gene fusions involving the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, have been identified in 1-5% of lung cancers, suggesting that similar rare gene fusions may occur in other common epithelial cancers, including prostate cancer. Here we used paired-end transcriptome sequencing to screen ETS rearrangement-negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF (solute carrier family 45, member 3-v-raf murine sarcoma viral oncogene homolog B1) and ESRP1-RAF1 (epithelial splicing regulatory protein-1-v-raf-1 murine leukemia viral oncogene homolog-1) gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and mitogen-activated protein kinase kinase (MAP2K1) inhibitors. Screening a large cohort of patients, we found that, although rare, recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers and melanoma. Taken together, our results emphasize the key role of RAF family gene 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.

SLC45A3-ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer.

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 overexpression 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 harboring 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).

N-myc downstream regulated gene 1 (NDRG1) is fused to ERG in prostate cancer.

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.