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1.  The Role of SPINK1 in ETS Rearrangement Negative Prostate Cancers 
Cancer cell  2008;13(6):519-528.
Summary
ETS gene fusions have been characterized in a majority of prostate cancers, however the key molecular alterations in ETS negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers.
doi:10.1016/j.ccr.2008.04.016
PMCID: PMC2732022  PMID: 18538735
2.  mCOPA: analysis of heterogeneous features in cancer expression data 
Background
Cancer outlier profile analysis (COPA) has proven to be an effective approach to analyzing cancer expression data, leading to the discovery of the TMPRSS2 and ETS family gene fusion events in prostate cancer. However, the original COPA algorithm did not identify down-regulated outliers, and the currently available R package implementing the method is similarly restricted to the analysis of over-expressed outliers. Here we present a modified outlier detection method, mCOPA, which contains refinements to the outlier-detection algorithm, identifies both over- and under-expressed outliers, is freely available, and can be applied to any expression dataset.
Results
We compare our method to other feature-selection approaches, and demonstrate that mCOPA frequently selects more-informative features than do differential expression or variance-based feature selection approaches, and is able to recover observed clinical subtypes more consistently. We demonstrate the application of mCOPA to prostate cancer expression data, and explore the use of outliers in clustering, pathway analysis, and the identification of tumour suppressors. We analyse the under-expressed outliers to identify known and novel prostate cancer tumour suppressor genes, validating these against data in Oncomine and the Cancer Gene Index. We also demonstrate how a combination of outlier analysis and pathway analysis can identify molecular mechanisms disrupted in individual tumours.
Conclusions
We demonstrate that mCOPA offers advantages, compared to differential expression or variance, in selecting outlier features, and that the features so selected are better able to assign samples to clinically annotated subtypes. Further, we show that the biology explored by outlier analysis differs from that uncovered in differential expression or variance analysis. mCOPA is an important new tool for the exploration of cancer datasets and the discovery of new cancer subtypes, and can be combined with pathway and functional analysis approaches to discover mechanisms underpinning heterogeneity in cancers.
doi:10.1186/2043-9113-2-22
PMCID: PMC3553066  PMID: 23216803
Cancer; Outliers; Expression data; Expression profile; Cluster; Subtype; Heterogeneous; Bioinformatics; Percentile; Feature selection
3.  The novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under stress 
Cell death & disease  2010;1(4):e32-.
We previously identified a novel estrogen-induced gene, EIG121, as being differentially regulated in endometrioid and nonendometrioid endometrial carcinoma. The function of EIG121 was unknown. Using a tetracycline-inducible system, we found that overexpression of EIG121, but not of LacZ, caused a profound suppression of cell growth. Subcellular fractionation and immunofluroscent labeling indicated that EIG121 was a transmembrane protein localized in the plasma membrane-late endosome–lysosome compartments. Deletion of the putative transmembrane domain abolished the membrane association. In cells overexpressing EIG121, cytoplasmic vacuoles accumulated after EIG121 induction, and the autophagosome marker LC3 translocated into punctuate, dot-like structures. Electron microscopy revealed that in cells overexpressing EIG121, autophagosomes were markedly increased. Overexpression of EIG121 also increased the cells containing acidic vesicles and induced lysosomal degradation of long-lived proteins. In MCF-7 cells, both EIG121 and LC3 were rapidly degraded by a lysosomal mechanism after starvation. Knockdown of EIG121 blocked starvation-induced LC3 degradation. By itself, knockdown of EIG121 did not affect cell survival. When combined with starvation or cytotoxic agents, EIG121 knockdown greatly increased apoptosis. Our results suggest that EIG121 is associated with the endosome–lysosome compartments and may have an important role in autophagy. Under unfavorable conditions such as starvation and exposure to cytotoxic agents, EIG121 may protect cells from cell death by upregulating the autophagy pathway.
doi:10.1038/cddis.2010.9
PMCID: PMC2976047  PMID: 21072319
EIG121; autophagy; lysosome; starvation; apoptosis; cell survival
4.  The novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under stress 
Cell Death & Disease  2010;1(4):e32-.
We previously identified a novel estrogen-induced gene, EIG121, as being differentially regulated in endometrioid and nonendometrioid endometrial carcinoma. The function of EIG121 was unknown. Using a tetracycline-inducible system, we found that overexpression of EIG121, but not of LacZ, caused a profound suppression of cell growth. Subcellular fractionation and immunofluroscent labeling indicated that EIG121 was a transmembrane protein localized in the plasma membrane-late endosome–lysosome compartments. Deletion of the putative transmembrane domain abolished the membrane association. In cells overexpressing EIG121, cytoplasmic vacuoles accumulated after EIG121 induction, and the autophagosome marker LC3 translocated into punctuate, dot-like structures. Electron microscopy revealed that in cells overexpressing EIG121, autophagosomes were markedly increased. Overexpression of EIG121 also increased the cells containing acidic vesicles and induced lysosomal degradation of long-lived proteins. In MCF-7 cells, both EIG121 and LC3 were rapidly degraded by a lysosomal mechanism after starvation. Knockdown of EIG121 blocked starvation-induced LC3 degradation. By itself, knockdown of EIG121 did not affect cell survival. When combined with starvation or cytotoxic agents, EIG121 knockdown greatly increased apoptosis. Our results suggest that EIG121 is associated with the endosome–lysosome compartments and may have an important role in autophagy. Under unfavorable conditions such as starvation and exposure to cytotoxic agents, EIG121 may protect cells from cell death by upregulating the autophagy pathway.
doi:10.1038/cddis.2010.9
PMCID: PMC2976047  PMID: 21072319
EIG121; autophagy; lysosome; starvation; apoptosis; cell survival
5.  A Two-Step Toward Personalized Therapies for Prostate Cancer 
Science translational medicine  2011;3(72):72ps7.
Identifying the dominant genetic alterations that drive tumorigenesis is essential for developing targeted cancer therapies. Recent work has demonstrated that prostate tumors can be stratified by dominant genetic alterations, such as chromosomal rearrangements involving ETS (Erythroblastosis virus E26 transformation-specific) family transcription factors or overexpression of SPINK1, a gene that encodes a secreted serine protease inhibitor. In this issue of Science Translational Medicine, Ateeq et al. provide evidence to support a rationale for targeting the SPINK1 protein in the SPINK1+/ETS− subset of prostate tumors and also describe a potential interaction of SPINK1 with epidermal growth factor receptor that could be an additional target for therapeutic intervention.
doi:10.1126/scitranslmed.3002169
PMCID: PMC3089975  PMID: 21368221
6.  Characterization of Transcriptional Changes in ERG Rearrangement-Positive Prostate Cancer Identifies the Regulation of Metabolic Sensors Such as Neuropeptide Y 
PLoS ONE  2013;8(2):e55207.
ERG gene rearrangements are found in about one half of all prostate cancers. Functional analyses do not fully explain the selective pressure causing ERG rearrangement during the development of prostate cancer. To identify transcriptional changes in prostate cancer, including tumors with ERG gene rearrangements, we performed a meta-analysis on published gene expression data followed by validations on mRNA and protein levels as well as first functional investigations. Eight expression studies (n = 561) on human prostate tissues were included in the meta-analysis. Transcriptional changes between prostate cancer and non-cancerous prostate, as well as ERG rearrangement-positive (ERG+) and ERG rearrangement-negative (ERG−) prostate cancer, were analyzed. Detailed results can be accessed through an online database. We validated our meta-analysis using data from our own independent microarray study (n = 57). 84% and 49% (fold-change>2 and >1.5, respectively) of all transcriptional changes between ERG+ and ERG− prostate cancer determined by meta-analysis were verified in the validation study. Selected targets were confirmed by immunohistochemistry: NPY and PLA2G7 (up-regulated in ERG+ cancers), and AZGP1 and TFF3 (down-regulated in ERG+ cancers). First functional investigations for one of the most prominent ERG rearrangement-associated genes - neuropeptide Y (NPY) - revealed increased glucose uptake in vitro indicating the potential role of NPY in regulating cellular metabolism. In summary, we found robust population-independent transcriptional changes in prostate cancer and first signs of ERG rearrangements inducing metabolic changes in cancer cells by activating major metabolic signaling molecules like NPY. Our study indicates that metabolic changes possibly contribute to the selective pressure favoring ERG rearrangements in prostate cancer.
doi:10.1371/journal.pone.0055207
PMCID: PMC3563644  PMID: 23390522
7.  Distinct Genomic Aberrations Associated With ERG Rearranged Prostate Cancer 
Genes, chromosomes & cancer  2009;48(4):366-380.
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.
doi:10.1002/gcc.20647
PMCID: PMC2674964  PMID: 19156837
ETS genes; prostate cancer; gain; loss
8.  ETS gene fusions and prostate cancer 
Chromosomal rearrangements are common genetic alterations in solid tumors and hematologic neoplasias. Recently, gene fusions between erythroblastosis virus E26 transforming sequence (ETS) family of transcription factors and androgen-regulated, prostate-specific TMPRSS2 gene were detected in about 50% of prostate cancers. Further studies have shown a diversity of TMPRSS2:ETS hybrid transcripts and heterogeneity of the fusion genes in multifocal prostate cancer. The role of these gene fusions in prostate carcinogenesis, the protein products associated with the variant fusion transcripts and their association with tumor morphology, stage, and clinical outcomes have also been studied. Additional data have demonstrated ETS gene fusions as a potential biomarker for diagnosing and stratifying prostate cancer patients. The following summarizes these recent advances.
PMCID: PMC2780040  PMID: 19956446
Prostate cancer; ETS; TMPRSS2; gene fusion
9.  ERG rearrangement is specific to prostate cancer and does not occur in any other common tumor 
Background
Identification of specific somatic gene alterations is crucial for the insight into the development, progression, and clinical behavior of individual cancer types. The recently discovered recurrent ERG rearrangement in prostate cancer (PCa) might represent a PCa specific alteration that has not been systematically assessed in tumors other than PCa. Aim of this study was to assess, whether the ERG rearrangement and the distinct deletion site between TMPRSS2 and ERG, both predominantly resulting in a TMPRSS2-ERG fusion, occurs in tumors other than PCa.
Design
We assessed 54 different tumor types (2942 samples in total) for their ERG rearrangement status by FISH. To calibrate, we analyzed 285 PCa samples for the ERG rearrangement frequency. Additionally, we interrogated a high-resolution SNP data set across 3131 cancer specimens (26 tumor types) for copy number alterations.
Results
None of the 54 different tumor types assessed by FISH harbored an ERG rearrangement, whereas the PCa samples revealed an ERG rearrangement in 31.2%–49.5%, depending on the cohort. Furthermore, within the 26 tumor types assessed for copy number alterations by SNP, the distinct deletion site between TMPRSS2 and ERG (21q22.2-3) was detectable exclusively in PCa.
Conclusion
Although Ewing's sarcoma and AML have known rearrangements rarely involving ERG, we hypothesize that the ERG rearrangement as well as the distinct deletion site on 21q22.2-3 between TMPRSS2 and ERG, are PCa specific genomic alterations. These observations provide further insight into the oncogenesis of PCa and might be critical for the development of ERG rearrangement assessment as a clinical tool.
doi:10.1038/modpathol.2010.87
PMCID: PMC3606550  PMID: 20473283
ERG rearrangement; prostate cancer; carcinoma
10.  Prevalence of TMPRSS2-ERG and SLC45A3-ERG gene fusions in a large prostatectomy cohort 
The majority of prostate cancers harbor recurrent gene fusions between the hormone-regulated TMPRSS2 and members of the ETS family of transcription factors, most commonly ERG. Prostate cancer with ERG rearrangements represent a distinct subclass of tumor based on studies reporting associations with histomorphologic features, characteristic somatic copy number alterations, and gene expression signatures. The current study describes the frequency of ERG rearrangement prostate cancer and three 5 prime (5') gene fusion partners (i.e., TMPRSS2, SLC45A3 and NDRG1) in a large prostatectomy cohort.
ERG gene rearrangements and mechanism of rearrangement, as well as rearrangements of TMPRSS2, SLC45A3, and NDRG1 were assessed using fluorescence in-situ hybridization (FISH) on prostate cancer samples from 614 patients treated by radical prostatectomy. ERG rearrangement occurred in 53% of the 540 assessable cases. TMPRSS2 and SLC45A3 were the only 5' partner in 78% and 6% of these ERG rearranged cases, respectively. Interestingly, 11% of the ERG rearranged cases demonstrated concurrent TMPRSS2 and SLC45A3 rearrangements. TMPRSS2 or SLC45A3 rearrangements could not be identified for 5% of the ERG rearranged cases. From these remaining cases we identified one case with NDRG1 rearrangement. We did not observe any associations with pathologic parameters or clinical outcome.
This is the first study to describe the frequency of SLC45A3-ERG fusions in a large clinical cohort. Most studies have assumed that all ERG rearrangement prostate cancers harbor TMPRSS2-ERG fusions. This is also the first study to report concurrent TMPRSS2 and SLC45A3 rearrangements in the same tumor focus suggesting additional complexity that had not been previously appreciated. This study has important clinical implications for the development of diagnostic assays to detect ETS rearrangement prostate cancer. Incorporation of these less common ERG rearrangement prostate cancer fusion assays could further increase the sensitivity of these PCR-based approaches.
doi:10.1038/modpathol.2009.193
PMCID: PMC2848699  PMID: 20118910
Prostate cancer; ETS rearrangements; prevalence
11.  ERG Cooperates with Androgen Receptor in Regulating Trefoil Factor 3 in Prostate Cancer Disease Progression123 
Neoplasia (New York, N.Y.)  2010;12(12):1031-1040.
To elucidate the role of ETS gene fusions in castration-resistant prostate cancer (CRPC), we characterized the transcriptome of 54 CRPC tumor samples from men with locally advanced or metastatic disease. Trefoil factor 3 (TFF3) emerged as the most highly differentially regulated gene with respect to ERG rearrangement status and resistance to hormone ablation therapy. Conventional chromatin immunoprecipitation (ChIP)-polymerase chain reaction and ChIP followed by DNA sequencing (ChIP-seq) revealed direct binding of ERG to ETS binding sites in the TFF3 promoter in ERG-rearranged prostate cancer cell lines. These results were confirmed in ERG-rearranged hormone-naive prostate cancer (HNPC) and CRPC tissue samples. Functional studies demonstrated that ERG has an inhibitory effect on TFF3 expression in hormone-naive cancer but not in the castration-resistant state. In addition, we provide evidence suggesting an effect of androgen receptor signaling on ERG-regulated TFF3 expression. Furthermore, TFF3 overexpression enhances ERG-mediated cell invasion in CRPC prostate cancer cells. Taken together, our findings reveal a novel mechanism for enhanced tumor cell aggressiveness resulting from ERG rearrangement in the castration-resistant setting through TFF3 gene expression.
PMCID: PMC3003138  PMID: 21170267
12.  TopHat-Fusion: an algorithm for discovery of novel fusion transcripts 
Genome Biology  2011;12(8):R72.
TopHat-Fusion is an algorithm designed to discover transcripts representing fusion gene products, which result from the breakage and re-joining of two different chromosomes, or from rearrangements within a chromosome. TopHat-Fusion is an enhanced version of TopHat, an efficient program that aligns RNA-seq reads without relying on existing annotation. Because it is independent of gene annotation, TopHat-Fusion can discover fusion products deriving from known genes, unknown genes and unannotated splice variants of known genes. Using RNA-seq data from breast and prostate cancer cell lines, we detected both previously reported and novel fusions with solid supporting evidence. TopHat-Fusion is available at http://tophat-fusion.sourceforge.net/.
doi:10.1186/gb-2011-12-8-r72
PMCID: PMC3245612  PMID: 21835007
13.  The Mutational Landscape of Lethal Castrate Resistant Prostate Cancer 
Nature  2012;487(7406):239-243.
Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.
doi:10.1038/nature11125
PMCID: PMC3396711  PMID: 22722839
14.  Therapeutic targeting of SPINK1-positive prostate cancer 
Science translational medicine  2011;3(72):72ra17.
The discovery of recurrent gene fusions involving Erythroblastosis virus E26 transformation-specific (ETS) family transcription factors in approximately 50% of prostate cancers provides a basis for the molecular subclassification of prostate cancer. Previously, we showed that marked over-expression of SPINK1 (serine peptidase inhibitor, Kazal type 1), which encodes a secreted serine protease inhibitor, defines an aggressive molecular subtype of ETS fusion-negative prostate cancers (SPINK1+/ETS-, ~10% of all prostate cancers). Here, we examined the potential of SPINK1 as an extracellular therapeutic target in prostate cancer. We demonstrate that recombinant SPINK1 protein (rSPINK1) stimulates cell proliferation in benign RWPE and cancerous prostate cells. RWPE cells treated with rSPINK1 or conditioned medium from 22RV1 prostate cancer cells (SPINK1+/ETS-) showed significantly increased cell invasion and intravasation. Knockdown of SPINK1 in 22RV1 cells inhibited cell proliferation, cell invasion, and tumor growth in xenograft assays. Importantly, 22RV1 cell proliferation, invasion and intravasation were attenuated by an anti-SPINK1 monoclonal antibody (mAb). We also demonstrate that SPINK1 partially mediates its neoplastic effects through interaction with the epidermal growth factor receptor (EGFR). Administration of anti-SPINK1 mAb or anti-EGFR mAb (cetuximab) to mice bearing 22RV1 xenografts attenuated tumor growth by over 60% and 40% alone, respectively, and approximately 75% when combined, without affecting PC3 xenograft (SPINK1-/ETS-) growth. Taken together, this study qualifies SPINK1 as a therapeutic target in a subset of patients with SPINK1+/ETS- prostate cancer. Similar to antibody targeting of ERBB2 in a subset of breast cancers, our results provide rationale for both the development of humanized anti-SPINK1 monoclonal antibodies and evaluation of EGFR inhibition in SPINK1+/ETS- prostate cancers.
doi:10.1126/scitranslmed.3001498
PMCID: PMC3211047  PMID: 21368222
15.  Novel 5′ Fusion Partners of ETV1 and ETV4 in Prostate Cancer12 
Neoplasia (New York, N.Y.)  2013;15(7):720-726.
Gene fusions involving the erythroblast transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, ETV5, and FLI1 are a common feature of prostate carcinomas (PCas). The most common upstream fusion partner described is the androgen-regulated prostate-specific gene TMPRSS2, most frequently with ERG, but additional 5′ fusion partners have been described. We performed 5′ rapid amplification of cDNA ends in 18 PCas with ETV1, ETV4, or ETV5 outlier expression to identify the 5′ fusion partners. We also evaluated the exon-level expression profile of these ETS genes in 14 cases. We identified and confirmed by fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction the two novel chimeric genes OR51E2-ETV1 and UBTF-ETV4 in two PCas. OR51E2 encodes a G-protein-coupled receptor that is overexpressed in PCas, whereas UBTF is a ubiquitously expressed gene encoding an HMG-box DNA-binding protein involved in ribosome biogenesis. We additionally describe two novel gene fusion combinations of previously described genes, namely, SLC45A3-ETV4 and HERVK17-ETV4. Finally, we found one PCa with TMPRSS2-ETV1, one with C15orf21-ETV1, one with EST14-ETV1, and two with 14q133-q21.1-ETV1. In nine PCas (eight ETV1 and one ETV5), exhibiting ETS outlier expression and genomic rearrangement detected by FISH, no 5′ fusion partner was found. Our findings contribute significantly to characterize the heterogeneous group of ETS gene fusions and indicate that all genes described as 5′ fusion partners with one ETS gene can most likely be rearranged with any of the other ETS genes involved in prostate carcinogenesis.
PMCID: PMC3689235  PMID: 23814484
16.  Gene rearrangements in hormone receptor negative breast cancers revealed by mate pair sequencing 
BMC Genomics  2013;14:165.
Background
Chromosomal rearrangements in the form of deletions, insertions, inversions and translocations are frequently observed in breast cancer genomes, and a subset of these rearrangements may play a crucial role in tumorigenesis. To identify novel somatic chromosomal rearrangements, we determined the genome structures of 15 hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing.
Results
We identified and validated 40 somatic structural alterations, including the recurring fusion between genes DDX10 and SKA3 and translocations involving the EPHA5 gene. Other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. RNA interference-mediated suppression of five candidate genes (DDX10, SKA3, EPHA5, CLTC and TNIK) led to inhibition of breast cancer cell growth. Moreover, downregulation of DDX10 in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology.
Conclusions
Using whole genome mate pair sequencing and RNA interference assays, we have discovered a number of novel gene rearrangements in breast cancer genomes and identified DDX10, SKA3, EPHA5, CLTC and TNIK as potential cancer genes with impact on the growth and proliferation of breast cancer cells.
doi:10.1186/1471-2164-14-165
PMCID: PMC3600027  PMID: 23496902
17.  Characterization of TMPRSS2-ETS Gene Aberrations in Androgen Independent Metastatic Prostate Cancer 
Cancer research  2008;68(10):3584-3590.
Recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factor family members ERG, ETV1, and ETV4 have been identified as a critical event in prostate cancer development. In this study, we characterized the prevalence and diversity of these rearrangements in hormone-refractory metastatic prostate cancer. We employed a fluorescence in situ hybridization (FISH) split probe strategy to comprehensively evaluate TMPRSS2: ETS aberrations across 97 non-osseous metastatic sites of prostate cancer from 30 rapid autopsies of men who died of androgen independent disease. Tissue microarrays were constructed representing multiple metastatic sites from each patient, and split signal FISH probes for TMPRSS2, ERG, ETV1 and ETV4 were used to assess for TMPRSS2-ETS rearrangements. In patients exhibiting these aberrations, multiple sites from an individual case harbored the same gene fusion molecular sub-type suggesting clonal expansion of disease. The most common prostate cancer gene fusion, TMPRSS2-ERG, can be generated by the mechanism of intrachromosomal deletion (Edel) about 39–60% of the time in clinically localized disease (1, 2). Interestingly, we observed that all of the androgen independent metastatic prostate cancer sites harboring TMPRSS2-ERG were associated with Edel. These findings suggest that TMPRSS2-ERG with Edel is an aggressive, and in this study uniformly lethal, molecular sub-type of prostate cancer associated with androgen-independent disease.
doi:10.1158/0008-5472.CAN-07-6154
PMCID: PMC2677168  PMID: 18483239
18.  State of art fusion-finder algorithms are suitable to detect transcription-induced chimeras in normal tissues? 
BMC Bioinformatics  2013;14(Suppl 7):S2.
Background
RNA-seq has the potential to discover genes created by chromosomal rearrangements. Fusion genes, also known as "chimeras", are formed by the breakage and re-joining of two different chromosomes. It is known that chimeras have been implicated in the development of cancer. Few publications in the past showed the presence of fusion events also in normal tissue, but with very limited overlaps between their results. More recently, two fusion genes in normal tissues were detected using both RNA-seq and protein data.
Due to heterogeneous results in identifying chimeras in normal tissue, we decided to evaluate the efficacy of state of the art fusion finders in detecting chimeras in RNA-seq data from normal tissues.
Results
We compared the performance of six fusion-finder tools: FusionHunter, FusionMap, FusionFinder, MapSplice, deFuse and TopHat-fusion. To evaluate the sensitivity we used a synthetic dataset of fusion-products, called positive dataset; in these experiments FusionMap, FusionFinder, MapSplice, and TopHat-fusion are able to detect more than 78% of fusion genes. All tools were error prone with high variability among the tools, identifying some fusion genes not present in the synthetic dataset. To better investigate the false discovery chimera detection rate, synthetic datasets free of fusion-products, called negative datasets, were used. The negative datasets have different read lengths and quality scores, which allow detecting dependency of the tools on both these features. FusionMap, FusionFinder, mapSplice, deFuse and TopHat-fusion were error-prone. Only FusionHunter results were free of false positive. FusionMap gave the best compromise in terms of specificity in the negative dataset and of sensitivity in the positive dataset.
Conclusions
We have observed a dependency of the tools on read length, quality score and on the number of reads supporting each chimera. Thus, it is important to carefully select the software on the basis of the structure of the RNA-seq data under analysis. Furthermore, the sensitivity of chimera detection tools does not seem to be sufficient to provide results consistent with those obtained in normal tissues on the basis of fusion events extracted from published data.
doi:10.1186/1471-2105-14-S7-S2
PMCID: PMC3633050  PMID: 23815381
19.  A Novel 4-color Fluorescence in Situ Hybridization Assay for Detection of TMPRSS2 and ERG Rearrangements in Prostate Cancer 
Cancer genetics  2013;206(0):1-11.
Purpose
Since the identification of TMPRSS2/ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We hereby report a novel 4-color fluorescence in situ hybridization (FISH) assay that not only detects the typical TMPRSS2:ERG fusion but also alternative rearrangements of either the TMPRSS2 or ERG gene.
Experimental design
We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens including cell lines, primary prostate cancer, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients.
Results
When compared with RT-PCR or Gen-Probe method as the technical reference, the 4-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% Confidence Interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting TMPRSS2:ERG fusion. TMPRSS2:ERG fusion was detected at 41% and 43% in primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Alternative rearrangements other than the typical TMPRSS2:ERG fusion were confirmed by karyotype analysis and shown present in 7% primary cancer and 13% CRPC tumors. Successful karyotype analysis is reported for the first time on four of the xenograft samples, complementing the FISH results.
Conclusions
This 4-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer.
doi:10.1016/j.cancergen.2012.12.004
PMCID: PMC3632390  PMID: 23352841
Prostate cancer; TMPRSS2; ERG; FISH
20.  Recurrent Rearrangements in Prostate Cancer: Causes and Therapeutic Potential 
Current drug targets  2013;14(4):450-459.
DNA damage and genetic rearrangements are hallmarks of cancer. However, gene fusions as driver mutations in cancer have classically been a distinction in leukemia and other rare instances until recently with the discovery of gene fusion events occurring in 50 to 75% of prostate cancer patients. The discovery of the TMPRSS2-ERG fusion sparked an onslaught of discovery and innovation resulting in a delineation of prostate cancer via a molecular signature of gene fusion events. The increased commonality of high-throughput sequencing data coupled with improved bioinformatics approaches not only elucidated the molecular underpinnings of prostate cancer progression, but the mechanisms of gene fusion biogenesis. Interestingly, the androgen receptor (AR), already known to play a significant role in prostate cancer tumorigenesis, has recently been implicated in the processes resulting in gene fusions by inducing the spatial proximity of genes involved in rearrangements, promoting the formation of double-strand DNA breaks (DSB), and facilitating the recruitment of proteins for non-homologous end-joining (NHEJ). Our increased understanding of the mechanisms inducing genomic instability may lead to improved diagnostic and therapeutic strategies. To date, the majority of prostate cancer patients can be molecularly stratified based on their gene fusion status thereby increasing the potential for tailoring more specific and effective therapies.
PMCID: PMC3733264  PMID: 23410129
Androgen receptor; gene fusions; genomic instability; prostate cancer
21.  Chromosome rearrangement associated inactivation of tumour suppressor genes in prostate cancer 
Prostate cancer, the most common male cancer in Western countries, is commonly detected with complex chromosomal rearrangements. Following the discovery of the recurrent TMPRSS2:ETS fusions in prostate cancer and EML4:ALK in non-small-cell lung cancer, it is now accepted that fusion genes not only are the hallmark of haematological malignancies and sarcomas, but also play an important role in epithelial cell carcinogenesis. However, previous studies aiming to identify fusion genes in prostate cancer were mainly focused on expression changes and fusion transcripts. To investigate the genes recurrently affected by the chromosome breakpoints in prostate cancer, we analysed Affymetrix array 6.0 and 500K SNP microarray data from 77 prostate cancer samples. While the two genes most frequently affected by genomic breakpoints were, as expected, ERG and TMPRSS2, surprisingly more known tumour suppressor genes (TSGs) than known oncogenes were identified at recurrent chromosome breakpoints. Certain well-characterised TSGs, including p53, PTEN, BRCA1 and BRCA2 are recurrently truncated as a result of chromosome rearrangements in prostate cancer. Interestingly, many of the genes residing at recurrent breakpoint sites have not yet been implicated in prostate carcinogenesis such as HOOK3, PPP2R2A and TCBA1. We have confirmed the generally reduced expression of selected genes in clinical samples using quantitative RT-PCR analysis. Subsequently, we further investigated the genes associated with the t(4:6) translocation in LNCaP cells and reveal the genomic fusion of SNX9 and putative TSG UNC5C, which led to the reduced expression of both genes. This study reveals another common mechanism that leads to the inactivation of TSGs in prostate cancer and the identification of multiple TSGs inactivated by chromosome rearrangements will lead to new direction of research for the molecular basis of prostate carcinogenesis.
PMCID: PMC3189822  PMID: 21994901
prostate cancer; chromosome rearrangements; chromosome breakpoints; tumour suppressor gene; oncogene; SNP array; FISH; QRT-PCR
22.  Functionally Recurrent Rearrangements of the MAST Kinase and Notch Gene Families in Breast Cancer 
Nature Medicine  2011;17(12):1646-1651.
Breast cancer is a heterogeneous disease, exhibiting a wide range of molecular aberrations and clinical outcomes. Here we employed paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers harbor an array of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving microtubule associated serine-threonine kinase (MAST) and Notch family genes. Both MAST and Notch family gene fusions exerted significant phenotypic effects in breast epithelial cells. Breast cancer lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and over-expression of MAST1 or MAST2 gene fusions had a proliferative effect both in vitro and in vivo. These findings illustrate that recurrent gene rearrangements play significant roles in subsets of carcinomas and suggest that transcriptome sequencing may serve to identify patients with rare, actionable gene fusions.
doi:10.1038/nm.2580
PMCID: PMC3233654  PMID: 22101766
23.  A fluorescence in situ hybridization screen for E26 transformation-specific aberrations: identification of DDX5-ETV4 fusion protein in prostate cancer 
Cancer research  2008;68(18):7629-7637.
Recurrent gene fusions involving ETS transcription factors ERG, ETV1, ETV4, or ETV5 have been identified in 40–70% of prostate cancers. Here we employed a comprehensive fluorescence in situ hybridization (FISH) split probe strategy interrogating all 27 ETS family members and their five known 5′ fusion partners in a cohort of 110 clinically localized prostate cancer patients. Gene rearrangements were only identified in ETS genes that were previously implicated in prostate cancer gene fusions including ERG, ETV1, and ETV4 (43%, 5% and 5%, respectively), suggesting that a substantial fraction of prostate cancers (estimated at 30–60%) cannot be attributed to an ETS gene fusion. Among the known 5′ gene fusion partners, TMPRSS2 was rearranged in 47% of cases followed by SLC45A3, HNRPA2B1, and C15ORF21 in 2%, 1% and 1% of cases, respectively. Based on this comprehensive FISH screen, we have made four noteworthy observations. First, by screening the entire ETS transcription factor family for rearrangements, we found that a large fraction of prostate cancers (44%) cannot be ascribed to an ETS gene fusion, an observation which will stimulate research into identifying recurrent non-ETS aberrations in prostate cancers. Second, we identified SLC45A3 as a novel 5′ fusion partner of ERG; previously, TMPRSS2 was the only described 5′ partner of ERG. Third, we identified two prostate-specific, androgen-induced genes, FLJ37254 and CANT1 as 5′ partners to ETV1. And fourth, we identified a ubiquitously expressed, androgen-insensitive gene DDX5 fused in frame with ETV4 leading to the expression of a DDX5:ETV4 fusion protein.
doi:10.1158/0008-5472.CAN-08-2014
PMCID: PMC2760292  PMID: 18794152
gene fusion; prostate cancer; ETS; DDX5; ETV4; fusion protein
24.  LSOSS: Detection of Cancer Outlier Differential Gene Expression 
Biomarker Insights  2010;5:69-78.
Detection of differential gene expression using microarray technology has received considerable interest in cancer research studies. Recently, many researchers discovered that oncogenes may be activated in some but not all samples in a given disease group. The existing statistical tools for detecting differentially expressed genes in a subset of the disease group mainly include cancer outlier profile analysis (COPA), outlier sum (OS), outlier robust t-statistic (ORT) and maximum ordered subset t-statistics (MOST). In this study, another approach named Least Sum of Ordered Subset Square t-statistic (LSOSS) is proposed. The results of our simulation studies indicated that LSOSS often has more power than previous statistical methods. When applied to real human breast and prostate cancer data sets, LSOSS was competitive in terms of the biological relevance of top ranked genes. Furthermore, a modified hierarchical clustering method was developed to classify the heterogeneous gene activation patterns of human breast cancer samples based on the significant genes detected by LSOSS. Three classes of gene activation patterns, which correspond to estrogen receptor (ER)+, ER− and a mixture of ER+ and ER−, were detected and each class was assigned a different gene signature.
PMCID: PMC2918352  PMID: 20703321
differential gene expression; cancer; outlier
25.  TMPRSS2-ERG gene fusion is not associated with outcome in patients treated by prostatectomy 
Cancer research  2009;69(4):1400-1406.
A significant number of prostate cancers have been shown to have recurrent chromosomal rearrangements resulting in the fusion of the androgen regulated TMPRSS2 promoter to a member of the ETS transcription factor family, most commonly ERG. This results in ERG overexpression which may have a direct causal role in prostate tumorigenesis or progression. However, the clinical significance of the rearrangement is unclear and, in particular, relationship to outcome has been inconsistent in recent reports. We analyzed TMPRSS2-ERG gene rearrangement status by fluorescence in situ hybridization (FISH) in 521 cases of clinically localized surgically treated prostate cancer with 95 months median follow-up and also in 40 unmatched metastases. 42% of primary tumors and 40% of metastases had rearrangements. 11% had copy number increase (CNI) of the TMPRRS2-ERG region. Rearrangement alone was associated with lower grade, but not with stage, biochemical recurrence, metastases or death. CNI with and without rearrangement was associated with high grade and advanced stage. Further, a subgroup of cancers with CNI and rearrangement by deletion, with two or more copies of the deleted locus, tended to be more clinically aggressive. DNA index assessment revealed that the majority of tumors with CNI of TMPRSS2-ERG had generalized aneuploidy/ tetraploidy in contrast to tumors without TMPRSS2-ERG CNI, which were predominantly diploid. We therefore conclude that translocation of TMPRSS2-ERG is not associated with outcome and the aggressive clinical features associated with CNI of chromosome 21 reflect generalized aneuploidy and are not due to CNI specifically of rearranged TMPRSS2-ERG.
doi:10.1158/0008-5472.CAN-08-2467
PMCID: PMC3676271  PMID: 19190343
TMPRSS2; ERG; fusion; prostate; outcome

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