Quantitative targeted proteomics has recently taken front stage in the proteomics community. Centered on multiple reaction monitoring–mass spectrometry (MRM–MS) methodologies, quantitative targeted proteomics is being used in the verification of global proteomics data, the discovery of lower abundance proteins, protein post-translational modifications, discrimination of select highly homologous protein isoforms and as the final step in biomarker discovery. An older methodology utilized with small molecule analysis, the proteomics community is making great technological strides to develop MRM–MS as the next method to address previously challenging issues in global proteomics experimentation, namely dynamic range, identification of post-translational modifications, sensitivity and selectivity of measurement which will undoubtedly further biomedical knowledge. This brief review will provide a general introduction of MRM–MS and highlight its novel application for targeted quantitative proteomic experimentations.

Abstract

Apoptosis is a fundamental biologic process by which metazoan cells orchestrate their own self-demise. Genetic analyses of the nematode C elegans identified three core components of the suicide apparatus which include CED-3, CED-4, and CED-9. An analogous set of core constituents exists in mammalian cells and includes caspase-9, Apaf-1, and bcl-2/xl, respectively. CED-3 and CED-4, along with their mammalian counterparts, function to kill cells, whereas CED-9 and its mammalian equivalents protect cells from death. These central components biochemically intermingle in a ternary complex recently dubbed the “apoptosome.” The C elegans protein EGL-1 and its mammalian counterparts, pro-apoptotic members of the bcl-2 family, induce cell death by disrupting apoptosome interactions. Thus, EGL-1 may represent a primordial signal integrator for the apoptosome. Various biochemical processes including oligomerization, adenosine triphosphate ATP/dATP binding, and cytochrome c interaction play a role in regulating the ternary death complex. Recent studies suggest that cell death receptors, such as CD95, may amplify their suicide signal by activating the apoptosome. These mutual associations by core components of the suicide apparatus provide a molecular framework in which diverse death signals likely interface. Understanding the apoptosome and its cellular connections will facilitate the design of novel therapeutic strategies for cancer and other disease states in which apoptosis plays a pivotal role.

Background

Despite significant advancement in alignment algorithms, the exponential growth of nucleotide sequencing throughput threatens to outpace bioinformatic analysis. Computation may become the bottleneck of genome analysis if growing alignment costs are not mitigated by further improvement in algorithms. Much gain has been gleaned from indexing and compressing alignment databases, but many widely used alignment tools process input reads sequentially and are oblivious to any underlying redundancy in the reads themselves.

Results

Here we present Oculus, a software package that attaches to standard aligners and exploits read redundancy by performing streaming compression, alignment, and decompression of input sequences. This nearly lossless process (> 99.9%) led to alignment speedups of up to 270% across a variety of data sets, while requiring a modest amount of memory. We expect that streaming read compressors such as Oculus could become a standard addition to existing RNA-Seq and ChIP-Seq alignment pipelines, and potentially other applications in the future as throughput increases.

Conclusions

Oculus efficiently condenses redundant input reads and wraps existing aligners to provide nearly identical SAM output in a fraction of the aligner runtime. It includes a number of useful features, such as tunable performance and fidelity options, compatibility with FASTA or FASTQ files, and adherence to the SAM format. The platform-independent C++ source code is freely available online, at http://code.google.com/p/oculus-bio.

High-resolution magic-angle spinning (HR-MAS) proton NMR spectroscopy is used to explore the metabolic signatures of head and neck squamous cell carcinoma (HNSCC) which included matched normal adjacent tissue (NAT) and tumor originating from tongue, lip, larynx and oral cavity, and associated lymph-node metastatic (LN-Met) tissues. A total of 43 tissues (18 NAT, 18 Tumor and 7 LN-Met) from twenty-two HNSCC patients were analyzed. Principal Component Analysis of NMR data showed a clear classification between NAT and tumor tissues, however, LN-Met tissues were classified among tumor. A partial least squares discriminant analysis model generated from NMR metabolic profiles was used to differentiate normal from tumor samples (Q2 > 0.80, Receiver Operator Characteristic area under the curve > 0. 86, using 7-fold cross validation). HNSCC and LN-Met tissues showed elevated levels of lactate, amino acids including leucine, isoleucine, valine, alanine, glutamine, glutamate, aspartate, glycine, phenylalanine and tyrosine, choline containing compounds, creatine, taurine, glutathione and decreased levels of triglycerides. These elevated metabolites were associated with highly active glycolysis, increased amino acids influx (anaplerosis) into the TCA cycle, altered energy metabolism, membrane choline phospholipid metabolism, and oxidative and osmotic defense mechanisms. Moreover, decreased levels of triglycerides may indicate lipolysis followed by β-oxidation of fatty acids that may exist to deliver bioenergy for rapid tumor cell proliferation and growth.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that most commonly evolves from preexisting prostate adenocarcinoma (PCA). Using Next Generation RNA-sequencing and oligonucleotide arrays, we profiled 7 NEPC, 30 PCA, and 5 benign prostate tissue (BEN), and validated findings on tumors from a large cohort of patients (37 NEPC, 169 PCA, 22 BEN) using IHC and FISH. We discovered significant overexpression and gene amplification of AURKA and MYCN in 40% of NEPC and 5% of PCA, respectively, and evidence that that they cooperate to induce a neuroendocrine phenotype in prostate cells. There was dramatic and enhanced sensitivity of NEPC (and MYCN overexpressing PCA) to Aurora kinase inhibitor therapy both in vitro and in vivo, with complete suppression of neuroendocrine marker expression following treatment. We propose that alterations in Aurora kinase A and N-myc are involved in the development of NEPC, and future clinical trials will help determine from the efficacy of Aurora kinase inhibitor therapy.

Summary: Next generation sequencing (NGS) technologies have enabled de novo gene fusion discovery that could reveal candidates with therapeutic significance in cancer. Here we present an open-source software package, ChimeraScan, for the discovery of chimeric transcription between two independent transcripts in high-throughput transcriptome sequencing data.

Availability: http://chimerascan.googlecode.com

Contact: cmaher@dom.wustl.edu

Supplementary Information: Supplementary data are available at Bioinformatics online.

Ewing's sarcoma family tumors (ESFTs) are aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here we report that these fusion products interact with the DNA damage response protein and transcriptional co-regulator PARP-1. ESFT cells, primary tumor xenografts and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1-driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI-mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1: PARP1 intersection as a therapeutic strategy to improve the treatment of Ewing's sarcoma family tumors.

Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are frequently altered in cancer. C-terminal binding protein 1 (CtBP1), a transcriptional corepressor that regulates the expression of tumor suppressors and genes involved in cell death, is known to play a role in multiple cancers. In this study, we observed the overexpression and mislocalization of CtBP1 in metastatic prostate cancer and demonstrated the functional significance of CtBP1 in prostate cancer progression. Transient and stable knockdown of CtBP1 in prostate cancer cells inhibited their proliferation and invasion. Expression profiling studies of prostate cancer cell lines revealed that multiple tumor suppressor genes are repressed by CtBP1. Furthermore, our studies indicate a role for CtBP1 in conferring radiation resistance to prostate cancer cell lines. In vivo studies using chicken chorioallantoic membrane assay, xenograft studies, and murine metastasis models suggested a role for CtBP1 in prostate tumor growth and metastasis. Taken together, our studies demonstrated that dysregulated expression of CtBP1 plays an important role in prostate cancer progression and may serve as a viable therapeutic target.

Recurrent gene fusions involving ETS family genes are a distinguishing feature of human prostate cancers, with TMPRSS2-ERG fusions representing the most common subtype. The TMPRSS2-ERG fusion transcript and its splice variants are well characterized in prostate cancers, however not much is known about the levels and regulation of wild-type ERG. By employing an integrative approach, we demonstrate that the TMPRSS2-ERG gene fusion product binds to the ERG locus and drives the over-expression of wild-type ERG in prostate cancers. Knock-down of TMPRSS2-ERG in VCaP cells resulted in the down regulation of wild-type ERG transcription, while stable over-expression of TMPRSS2-ERG in the gene fusion-negative PC3 cells was associated with the up-regulation of wild-type ERG transcript. Further, androgen signaling-mediated up-regulation of TMPRSS2-ERG resulted in the concomitant up-regulation of wild-type ERG transcription in VCaP cells. The loss of wild-type ERG expression was associated with a decrease in the invasive potential of VCaP cells. Importantly, 38% of clinically localized prostate cancers and 27% of metastatic prostate cancers harboring the TMPRSS2-ERG gene fusions exhibited over-expression of wild-type ERG. Taken together, these results provide novel insights into the regulation of ERG in human prostate cancers.

Purpose

External beam radiation therapy is often used as in an attempt to cure localized prostate cancer (PCa), but is only palliative against disseminated disease. Raf Kinase Inhibitory Protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test if radiation therapy similarly induces apoptosis through induction of RKIP expression.

Methods

The C4-2B PCa cell line was engineered to over express or under express RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice following radiation treatment.

Results

Radiation induced both RKIP expression and apoptosis of PCa cells. Over expression of RKIP sensitized PCa cells to radiation-induced apoptosis; whereas, short-hairpin targeting of RKIP, so that radiation could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered upon manipulation of RKIP expression revealed that an inverse correlation with the concept of genes altered by irradiation.

Conclusion

The data presented here indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that loss of RKIP confers a growth advantage upon PCa cells at distant sites since loss of RKIP would decrease apoptosis, favoring proliferation.

Application of high-throughput transcriptome sequencing has spurred highly sensitive detection and discovery of gene fusions in cancer, but distinguishing potentially oncogenic fusions from random, “passenger” aberrations has proven challenging. Here we examine a distinctive group of gene fusions that involve genes present in the loci of chromosomal amplifications—a class of oncogenic aberrations that are widely prevalent in breast cancers. Integrative analysis of a panel of 14 breast cancer cell lines comparing gene fusions discovered by high-throughput transcriptome sequencing and genome-wide copy number aberrations assessed by array comparative genomic hybridization, led to the identification of 77 gene fusions, of which more than 60% were localized to amplicons including 17q12, 17q23, 20q13, chr8q, and others. Many of these fusions appeared to be recurrent or involved highly expressed oncogenic drivers, frequently fused with multiple different partners, but sometimes displaying loss of functional domains. As illustrative examples of the “amplicon-associated” gene fusions, we examined here a recurrent gene fusion involving the mediator of mammalian target of rapamycin signaling, RPS6KB1 kinase in BT-474, and the therapeutically important receptor tyrosine kinase EGFR in MDA-MB-468 breast cancer cell line. These gene fusions comprise a minor allelic fraction relative to the highly expressed full-length transcripts and encode chimera lacking the kinase domains, which do not impart dependence on the respective cells. Our study suggests that amplicon-associated gene fusions in breast cancer primarily represent a by-product of chromosomal amplifications, which constitutes a subset of passenger aberrations and should be factored accordingly during prioritization of gene fusion candidates.

Purpose

Bone is the most common metastatic site for prostate cancer, and osseous metastases are the leading cause of morbidity from this disease. Recent autopsy studies prove that 100% of men who die of prostate cancer have bone involvement. Understanding the biology of prostate cancer and its evolution to an incurable androgen independent phenotype requires an understanding of the genetic and cellular alterations that lead to the seeding and proliferation of tumor foci in bone, as well as the microenvironment in which these metastases arise. No intensive studies, however, have been conducted on osseous metastatic tissues from patients with metastatic prostate cancer due to lack of access to such tissues for profiling and other research.

Experimental Design

We demonstrate, for the first time, a reproducible methodology to obtain high quality clinical tumor tissues metastatic to the bone. This technique allowed the procurement of viable metastatic tumor tissue from involved bones in 13 recent autopsies conducted at the University of Michigan, and analyzed the gene expression of these tissues using real time PCR and microarrays.

Results

We present here the discovery of non-ossified bone metastases from multiple patients with advanced prostate cancer and their subsequent characterization and comparison to non-osseous metastases from the same patients

Conclusion

This represents a versatile and practical approach that may be employed to characterize the steps in metastasis and the phenotypic characteristics of osseous metastasis of prostate cancer and to profile RNA, DNA and cDNA from tumor samples metastatic to the bone.

Rap1GAP is a critical tumor suppressor gene that is down-regulated in multiple aggressive cancers such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP down-regulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb mediated repression of rap1GAP that involves EZH2, a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 up-regulation, and the concomitant down-regulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of H3K27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.

The discovery of numerous non-coding RNA (ncRNA) transcripts in species from yeast to mammals has dramatically altered our understanding of cell biology, especially disease biology such as cancer. In humans, the identification of abundant long ncRNA (lncRNAs) >200 bp in length has catalyzed their characterization as critical components of cancer biology. Recently, roles for lncRNAs as drivers of tumor suppressive and oncogenic functions have appeared in prevalent cancer types, such as breast and prostate cancer. In this review, we will highlight the emerging impact of ncRNAs in cancer research, with a particular focus on the mechanisms and functions of lncRNAs.

The theory of Darwinian evolution is the fundamental keystones of modern biology. Late in the last century, computer scientists began adapting its principles, in particular natural selection, to complex computational challenges, leading to the emergence of evolutionary algorithms. The conceptual model of selective pressure and recombination in evolutionary algorithms allows scientists to efficiently search high dimensional space for solutions to complex problems. In the last decade, genetic programming has been developed and extensively applied for analysis of molecular data to classify cancer subtypes and characterize the mechanisms of cancer pathogenesis and development. This article reviews current successes using genetic programming and discusses its potential impact in cancer research and treatment in the near future.

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.

The research community at large is expending considerable resources to sequence the coding region of the genomes of tumors and other human diseases using targeted exome capture (i.e., “whole exome sequencing”). The primary goal of targeted exome sequencing is to identify nonsynonymous mutations that potentially have functional consequences. Here, we demonstrate that whole-exome sequencing data can also be analyzed for comprehensively monitoring somatic copy number alterations (CNAs) by benchmarking the technique against conventional array CGH. A series of 17 matched tumor and normal tissues from patients with metastatic castrate-resistant prostate cancer was used for this assessment. We show that targeted exome sequencing reliably identifies CNAs that are common in advanced prostate cancer, such as androgen receptor (AR) gain and PTEN loss. Taken together, these data suggest that targeted exome sequencing data can be effectively leveraged for the detection of somatic CNAs in cancer.

Patients with advanced prostate cancer (PCa) are initially susceptible to androgen withdrawal (AW), but ultimately develop resistance to this therapy (castration-resistant PCa, CRPC). Here we show that AW can promote CRPC development by increasing the levels of the receptor tyrosine kinase (RTK) ErbB3 in androgen-dependent PCa, resulting in AW-resistant cell cycle progression and increased androgen receptor (AR) transcriptional activity. CRPC cell lines and human prostate cancer tissue overexpressed ErbB3, whereas downregulation of ErbB3 prevented CRPC cell growth. Investigation of the mechanism by which AW augments ErbB3, using normal prostate derived pRNS-1-1 cells, and androgen-dependent PCa lines LNCaP, PC346C and CWR22 mouse xenografts, revealed that the AR suppresses ErbB3 protein levels, while AW relieves this suppression, demonstrating for the first time negative regulation of ErbB3 by AR. We show that AR activation promotes ErbB3 degradation in androgen-dependent cells, and that this effect is mediated by AR-dependent transcriptional upregulation of Nrdp1, an E3 ubiquitin ligase that targets ErbB3 for degradation but whose role in PCa has not been previously examined. Therefore, AW decreases Nrdp1 expression, promoting ErbB3 protein accumulation, and leading to AR-independent proliferation. However, in CRPC sublines of LNCaP and CWR22 which strongly overexpress the AR, ErbB3 levels remain elevated due to constitutive suppression of Nrdp1, which prevents AR regulation of Nrdp1. Our observations point to a model of CRPC development where progression of PCa to castration-resistance is associated with the inability of AR to transcriptionally regulate Nrdp1, and predict that inhibition of ErbB3 during AW may impair CRPC development.

Purpose

Clinical benign prostatic hyperplasia (BPH) is primarily diagnosed based on a diverse array of progressive lower urinary tract symptoms (LUTS) and is likely distinct from histological BPH, which is detected by the presence of non-malignant proliferation of prostate cells but may or may not be associated with symptoms. Pharmacological management of LUTS has emerged as an effective initial treatment for clinical BPH due to the introduction of new drug therapies shown to be effective in recent large clinical trials. Despite advances in symptom management and research into BPH pathology, diagnostic strategies for prediction of BPH progression and response to drug modalities are lacking and questions remain as to the molecular differences underlying clinical (symptomatic) versus histological (non-symptomatic) BPH.

Materials and Methods

As part of the Medical Therapy of Prostatic Symptoms (MTOPS) clinical trial, which demonstrated the effectiveness of combination drug therapy in slowing BPH progression, an archive of biological specimens linked to clinical data were collected for future profiling of disease pathology and changes associated with response to drug therapy. The MTOPS Prostatic Samples Analysis (MPSA) Consortium was established to identify and validate molecular markers that may better define BPH-related pathologies, identify risk for progression of LUTS, and predict response to drug therapy, using this MTOPS archive. The cooperating MPSA Biomarker Discovery Sites and Pathology Coordinating Center employ diverse methodologies and scientific approaches and unique expertise in addressing the goals of the consortium.

Results

To date the MPSA has identified a number of promising biomarkers and other molecular and cellular changes associated with BPH.

Conclusions

These findings and ongoing consortium discovery efforts have the potential to provide a greater understanding of the defects underlying disease pathology and may lead to the development of early and more effective pharmacological treatment strategies for BPH.

Cancer genomes contain many aberrant gene fusions—a few that drive disease and many more that are nonspecific passengers. We developed an algorithm (the concept signature or ‘ConSig’ score) that nominates biologically important fusions from high-throughput data by assessing their association with ‘molecular concepts’ characteristic of cancer genes, including molecular interactions, pathways and functional annotations. Copy number data supported candidate fusions and suggested a breakpoint principle for intragenic copy number aberrations in fusion partners. By analyzing lung cancer transcriptome sequencing and genomic data, we identified a novel R3HDM2-NFE2 fusion in the H1792 cell line. Lung tissue microarrays revealed 2 of 76 lung cancer patients with genomic rearrangement at the NFE2 locus, suggesting recurrence. Knockdown of NFE2 decreased proliferation and invasion of H1792 cells. Together, these results present a systematic analysis of gene fusions in cancer and describe key characteristics that assist in new fusion discovery.

Background

The majority of prostate cancers harbor gene fusions of the 5′-untranslated region of the androgen-regulated transmembrane protease, serine 2 (TMPRSS2) promoter with erythroblast transformation specific (ETS) transcription factor family members. The common v-ets erythroblastosis virus E26 oncogene homolog [avian] (TMPRSS2–ERG) fusion is associated with a more aggressive clinical phenotype, implying the existence of a distinct subclass of prostate cancer defined by this fusion.

Methods

We used cDNA-mediated annealing, selection, ligation, and extension to determine the expression profiles of 6144 transcriptionally informative genes in archived biopsy samples from 455 prostate cancer patients in the Swedish Watchful Waiting cohort (1987–1999) and the US-based Physicians Health Study cohort (1983–2003). A gene expression signature for prostate cancers with the TMPRSS2-ERG fusion was determined using partitioning and classification models and used in computational functional analysis. Cell proliferation and TMPRSS2-ERG expression in androgen receptor–negative (NCI-H660) and –positive (VCaP-ERβ) prostate cancer cells after treatment with vehicle or estrogenic compounds were assessed by viability assays and quantitative polymerase chain reaction, respectively. All statistical tests were two-sided.

Results

We identified an 87-gene expression signature that distinguishes TMPRSS2-ERG fusion prostate cancer as a discrete molecular entity (area under the curve = 0.80, 95% confidence interval [CI] = 0.792 to 0.81; P<.001). Computational analysis suggested that this fusion signature was associated with estrogen receptor (ER) signaling. Viability of NCI-H660 cells decreased after treatment with estrogen (viability normalized to day 0, estrogen vs vehicle at day 8, mean = 2.04 vs 3.40, difference = 1.36, 95% CI = 1.12 to 1.62) or ERβ agonist (ERβ agonist vs vehicle at day 8, mean = 1.86 vs 3.40, difference = 1.54, 95% CI = 1.39 to 1.69) but increased after ERα agonist treatment (ERα agonist vs vehicle at day 8, mean = 4.36 vs 3.40, difference = 0.96, 95% CI = 0.68 to 1.23). Similarly, expression of TMPRSS2-ERG decreased after ERβ agonist treatment (fold change over internal control, ERβ agonist vs vehicle at 24 hours, NCI H660, mean = 0.57-fold vs 1.0-fold, difference = 0.43, 95% CI = 0.29-fold to 0.57-fold) and increased after ERα agonist treatment (ERα agonist vs vehicle at 24 hours, mean = 5.63-fold vs 1.0-fold, difference = 4.63-fold, 95% CI = 4.34-fold to 4.92-fold).

Conclusions

TMPRSS2-ERG fusion prostate cancer is a distinct molecular subclass. TMPRSS2-ERG expression is regulated by a novel ER-dependent mechanism.

The neuronal repellent SLIT2 is repressed in a number of cancer types primarily through promoter hypermethylation. SLIT2, however, has not been studied in prostate cancer. Through genome-wide location analysis we identified SLIT2 as a target of Polycomb group (PcG) protein EZH2. The EZH2-containing Polycomb repressive complexes bound to the SLIT2 promoter inhibiting its expression. SLIT2 was down-regulated in a majority of metastatic prostate tumors exhibiting a negative correlation with EZH2. This repressed expression could be restored by methylation inhibitors or EZH2-suppressing compounds. In addition, a low level of SLIT2 expression was associated with aggressive prostate, breast and lung cancers. Functional assays showed that SLIT2 inhibited prostate cancer cell proliferation and invasion. Thus, this study demonstrated for the first time epigenetic silencing of SLIT2 in prostate tumors, and supported SLIT2 as a potential biomarker for aggressive solid tumors. Importantly, PcG-mediated repression may serve as a precursor for the silencing of SLIT2 by DNA methylation in cancer.

Although prostate-specific antigen (PSA) serum level is currently the standard of care for prostate cancer screening in the United States, it lacks ideal specificity and additional biomarkers are needed to supplement or potentially replace serum PSA testing. Emerging evidence suggests that monitoring the noncoding RNA transcript PCA3 in urine may be useful in detecting prostate cancer in patients with elevated PSA levels. Here, we show that a multiplex panel of urine transcripts outperforms PCA3 transcript alone for the detection of prostate cancer. We measured the expression of seven putative prostate cancer biomarkers, including PCA3, in sedimented urine using quantitative PCR on a cohort of 234 patients presenting for biopsy or radical prostatectomy. By univariate analysis, we found that increased GOLPH2, SPINK1, and PCA3 transcript expression and TMPRSS2:ERG fusion status were significant predictors of prostate cancer. Multivariate regression analysis showed that a multiplexed model, including these biomarkers, outperformed serum PSA or PCA3 alone in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.758 for the multiplexed model versus 0.662 for PCA3 alone (P = 0.003). The sensitivity and specificity for the multiplexed model were 65.9% and 76.0%, respectively, and the positive and negative predictive values were 79.8% and 60.8%, respectively. Taken together, these results provide the framework for the development of highly optimized, multiplex urine biomarker tests for more accurate detection of prostate cancer.

Genomic translocations leading to the expression of chimeric transcripts characterize several hematologic, mesenchymal and epithelial malignancies. While several gene fusions have been linked to essential molecular events in hematologic malignancies, the identification and characterization of recurrent chimeric transcripts in epithelial cancers has been limited. However, the recent discovery of the recurrent gene fusions in prostate cancer has sparked a revitalization of the quest to identify novel rearrangements in epithelial malignancies. Here, the molecular mechanisms of gene fusions that drive several epithelial cancers and the recent technological advances that increase the speed and reliability of recurrent gene fusion discovery are explored.