Recurrent chromosomal rearrangements have not been well characterized in common carcinomas. We used a bioinformatics approach to discover candidate oncogenic chromosomal aberrations on the basis of outlier gene expression. Two ETS transcription factors, ERG and ETV1, were identified as outliers in prostate cancer. We identified recurrent gene fusions of the 5' untranslated region of TMPRSS2 to ERG or ETV1 in prostate cancer tissues with outlier expression. By using fluorescence in situ hybridization, we demonstrated that 23 of 29 prostate cancer samples harbor rearrangements in ERG or ETV1. Cell line experiments suggest that the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of ETS family members in prostate cancer. These results have implications in the development of carcinomas and the molecular diagnosis and treatment of prostate cancer.

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Prostate cancer is a common and clinically heterogeneous disease with marked variability in progression. The recent identification of gene fusions of the 5 ¶-untranslated region of TMPRSS2 (21q22.3) with the ETS transcription factor family members, either ERG (21q22.2), ETV1 (7p21.2), or ETV4 (17q21), suggests a mechanism for overexpression of the ETS genes in the majority of prostate cancers. In the current study using fluorescence in situ hybridization (FISH), we identified the TMPRSS2:ERG rearrangements in 49.2% of 118 primary prostate cancers and 41.2% of 18 hormone-naive lymph node metastases. The FISH assay detected intronic deletions between ERG and TMPRSS2 resulting in TMPRSS2:ERG fusion in 60.3% (35 of 58) of the primary TMPRSS2:ERG prostate cancers and 42.9% (3 of 7) of the TMPRSS2:ERG hormone-naive lymph node metastases. A significant association was observed between TMPRSS2:ERG rearranged tumors through deletions and higher tumor stage and the presence of metastatic disease involving pelvic lymph nodes. Using 100K oligonucleotide single nucleotide polymorphism arrays, a homogeneous deletion site between ERG and TMPRSS2 on chromosome 21q22.2-3 was identified with two distinct subclasses distinguished by the start point of the deletion at either 38.765 or 38.911 Mb. This study confirms that TMPRSS2:ERG is fused in approximately half of the prostate cancers through deletion of genomic DNA between ERG and TMPRSS2. The deletion as cause of TMPRSS2:ERG fusion is associated with clinical features for prostate cancer progression compared with tumors that lack the TMPRSS2:ERG rearrangement. (Cancer Res 2006; 66(17): 8337-41)

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.

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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).

Mutations in the androgen receptor (AR) that enable activation by antiandrogens occur in hormone-refractory prostate cancer, suggesting that mutant ARs are selected by treatment. To validate this hypothesis, we compared AR variants in metastases obtained by rapid autopsy of patients treated with flutamide or bicalutamide, or by excision of lymph node metastases from hormone-naïve patients. AR mutations occurred at low levels in all specimens, reflecting genetic heterogeneity of prostate cancer. Base changes recurring in multiple samples or multiple times per sample were considered putative selected mutations. Of 26 recurring missense mutations, most in the NH 2 -terminal domain (NTD) occurred in multiple tumors, whereas those in the ligand binding domain (LBD) were case specific. Hormone-naïve tumors had few recurring mutations and none in the LBD. Several AR variants were assessed for mechanisms that might underlie treatment resistance. Selection was evident for the promiscuous receptor AR-V716M, which dominated three metastases from one flutamide-treated patient. For the inactive cytoplasmically restricted splice variant AR23, coexpression with AR enhanced ligand response, supporting a decoy function. A novel NTD mutation, W435L, in a motif involved in intramolecular interaction influenced promoter-selective, cell-dependent transactivation. AR-E255K, mutated in a domain that interacts with an E3 ubiquitin ligase, led to increased protein stability and nuclear localization in the absence of ligand. Thus, treatment with antiandrogens selects for gain-of-function AR mutations with altered stability, promoter preference, or ligand specificity. These processes reveal multiple targets for effective therapies regardless of AR mutation. [Cancer Res 2009;69(10):4434-42]

The E-cadherin protein mediates Ca 2؉ -dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad 100 ) in prostate and mammary epithelial cells. Ecad 100 was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad 100 induction by ionomycin. Immunoblotting for spectrin and -calpain confirmed calpain activation in response to ionomycin treatment. Both the -and misoforms of calpain efficiently generated E-cad 100 in vitro. The E-cad 100 fragment was unable to bind to ␤-catenin, ␥-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the ␤-and ␥-catenin binding motifs of E-cadherin. Because Ecadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad 100 accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which Ecadherin is functionally inactivated through calpainmediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.The cadherin family of transmembrane glycoproteins mediates Ca 2ϩ -dependent intercellular adhesion in a homophilic manner (1). The classical cadherins, including N-, P-, and Ecadherin are well-conserved and are structurally similar. The cadherin structure consists of an extracellular domain with five tandem repeats, a transmembrane domain, and a cytoplasmic domain that interacts with the catenin family binding proteins (2). The interepithelial binding of E-cadherin, which mediates lateral cell-cell adhesion in secretory tissues such as the prostate and mammary gland (3), results in the formation of desmosomes and adherens junctions that are required for tissue morphogenesis and maintenance of the differentiated phenotype (1). The integrity of E-cadherin binding is dependent on its interaction with the catenin binding proteins in the intracellular domain. E-cadherin binds to ␤-, ␥-, and ␣-catenin (4), which physically associate with actin filaments and the actin cytoskeleton (5-7). Inactivation of E-cadherin through gene mutation, transcriptional inactivation, or promoter methylation has been demonstrated in many adenocarcinomas (8 -13). However, these mechanisms are not likely to account for all mechanisms by which E-cadherin becomes inactivated in cancer.One mechanis...