Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis. One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains. Here, we show the overexpression of P4HA1 in aggressive prostate cancer. Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression. Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression. Expression profiling studies using P4HA1-modulated prostate cells suggested regulation of Matrix metalloprotease 1. The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1. Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124. MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer. Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo. Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.
Prolyl 4-hydroxylase; alpha polypeptide I; Prostate Cancer; Progression; Metastasis; MicroRNA; Matrix metalloprotease 1
Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2 including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR gene fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Due to the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts which incorporate transcriptome analysis for gene fusions are poised to identify rare, targetable FGFR fusions across diverse cancer types.
MI-ONCOSEQ; integrative clinical sequencing; FGFR fusions; driver mutations; therapeutic targets
Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.
A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs all revealed the presence of a NAB2-STAT6 gene fusion. Using RT-PCR and sequencing, we detected this fusion in 51 of 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.
Histone methyltransferases (HMTases), as chromatin modifiers, regulate the transcriptomic landscape in normal development as well in diseases such as cancer. Here, we molecularly order two HMTases, EZH2 and MMSET that have established genetic links to oncogenesis. EZH2, which mediates histone H3K27 trimethylation and is associated with gene silencing, was shown to be coordinately expressed and function upstream of MMSET, which mediates H3K36 dimethylation and is associated with active transcription. We found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer.
Pseudogene transcripts can provide a novel tier of gene regulation through generation of endogenous siRNAs or miRNA-binding sites. Characterization of pseudogene expression, however, has remained confined to anecdotal observations due to analytical challenges posed by the extremely close sequence similarity with their counterpart coding genes. Here, we describe a systematic analysis of pseudogene “transcription” from an RNA-Seq resource of 293 samples, representing 13 cancer and normal tissue types, and observe a surprisingly prevalent, genome-wide expression of pseudogenes that could be categorized as ubiquitously expressed or lineage and/or cancer specific. Further, we explore disease subtype specificity and functions of selected expressed pseudogenes. Taken together, we provide evidence that transcribed pseudogenes are a significant contributor to the transcriptional landscape of cells and are positioned to play significant roles in cellular differentiation and cancer progression, especially in light of the recently described ceRNA networks. Our work provides a transcriptome resource that enables high-throughput analyses of pseudogene expression.
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.
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.
Polycomb Repressive Complexes (PRC1 and PRC2) mediated epigenetic regulation is critical for maintaining cellular homeostasis. Members of Polycomb Group (PcG) proteins including EZH2, a PRC2 component, are up-regulated in various cancer types, implicating their role in tumorigenesis. Here, we have identified several microRNAs (miRNAs) that are repressed by EZH2. These miRNAs in turn regulate the expression of PRC1 proteins, BMI1 and RING2. We found that ectopic overexpression of EZH2-regulated miRNAs attenuated cancer cell growth and invasiveness, and abrogated cancer stem cell properties. Importantly, expression analysis revealed an inverse correlation between miRNA and PRC protein levels in cell culture and prostate cancer tissues. Taken together, our data has uncovered a coordinate regulation of PRC1 and PRC2 activities that is mediated by miRNAs.
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.
ERG; prostate cancer; gene fusion
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.
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.
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
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.
Bone marrow; tumor; metastatic prostate cancer
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.
Recurrent fusions of ETS genes are considered driving mutations in a diverse array of cancers, including Ewing’s sarcoma, acute myeloid leukemia, and prostate cancer. We investigate the mechanisms by which ETS fusions mediate their effects, and find that the product of the predominant ETS gene fusion, TMPRSS2:ERG, interacts in a DNA-independent manner with the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and the catalytic subunit of DNA protein kinase (DNA-PKcs). ETS gene-mediated transcription and cell invasion require PARP1 and DNA-PKcs expression and activity. Importantly, pharmacological inhibition of PARP1 inhibits ETS positive, but not ETS negative, prostate cancer xenograft growth. Finally, overexpression of the TMPRSS2:ERG fusion induces DNA damage, which is potentiated by PARP1 inhibition in a manner similar to that of BRCA1/2-deficiency.
Prostate; Rearrangement; Gene Fusion; TMPRSS2; ERG; DNA-PKcs; PARP1
More than 1,000,000 men undergo prostate biopsy each year in the United States, most for “elevated” serum prostate specific antigen (PSA). Given the lack of specificity and unclear mortality benefit of PSA testing, methods to individualize management of elevated PSA are needed. Greater than 50% of PSA-screened prostate cancers harbor fusions between the transmembrane protease, serine 2 (TMPRSS2) and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) genes. Here, we report a clinical-grade, transcription-mediated amplification assay to risk stratify and detect prostate cancer noninvasively in urine. The TMPRSS2:ERG fusion transcript was quantitatively measured in prospectively collected whole urine from 1312 men at multiple centers. Urine TMPRSS2:ERG was associated with indicators of clinically significant cancer at biopsy and prostatectomy, including tumor size, high Gleason score at prostatectomy, and upgrading of Gleason grade at prostatectomy. TMPRSS2:ERG, in combination with urine prostate cancer antigen 3 (PCA3), improved the performance of the multivariate Prostate Cancer Prevention Trial risk calculator in predicting cancer on biopsy. In the biopsy cohorts, men in the highest and lowest of three TMPRSS2:ERG+PCA3 score groups had markedly different rates of cancer, clinically significant cancer by Epstein criteria, and high-grade cancer on biopsy. Our results demonstrate that urine TMPRSS2:ERG, in combination with urine PCA3, enhances the utility of serum PSA for predicting prostate cancer risk and clinically relevant cancer on biopsy.
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.
While chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers, how the fusion products regulate prostate cancer remains unclear. Using chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq), we found that ERG disrupts androgen receptor (AR) signaling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive epigenetic programs via direct activation of the H3K27 methyltransferase EZH2, a Polycomb group protein. These findings provide a working model in which TMPRSS2-ERG plays a critical role in cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the EZH2-mediated de-differentiation program.
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.
Polycomb group proteins; EZH2; SLIT2; prostate cancer; epigenetic silencing; DNA hypermethylation
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.
In bladder cancer, clinical grade and stage fail to capture outcome. We developed a clinically applicable quantitative polymerase chain reaction (QPCR) gene signature to predict progression in non-muscle-invasive bladder cancer. Comparative meta-profiling of twelve DNA microarray datasets (comprising 631 samples, 241,298 probe-sets) identified 96 genes which demonstrated differential expression in seven clinical outcome categories, or were identified as outliers, historic markers, or housekeeping genes. QPCR was performed to determine messenger RNA (mRNA) expression from 96 bladder tumors. 57 genes differentiated T2 from non-T2 tumors (p<0.05). Principal components analysis and Cox regression models were used to predict probability of T2 progression for non-T2 patients, placing them into high- and low-risk groups based on their gene expression. At two years, high-risk patients exhibited greater T2 progression (45% for high-risk patients vs. 12% for low-risk patients, p = 0.003, log-rank test). This difference remained significant within T1 (61% for high-risk vs. 22% for low-risk, p =0.02) and Ta tumors (29% for high-risk vs. 0% for low-risk, p=0.03). The best multivariate Cox model included stage and gender, and this signature provided predictive improvement over both (p=0.002, likelihood ratio test). Immunohistochemistry was performed for two genes in the signature not previously described in bladder cancer, ACTN1 (actinin) and CDC25B (cell division cycle 25B), corroborating their up-regulation at the protein level with disease progression. Thus, we identified a 57-gene QPCR panel to help predict progression of non-muscle-invasive bladder cancers and delineate a systematic, generalizable approach to converting microarray data into a multiplex assay for cancer progression.
urinary bladder neoplasms; disease progression; gene expression profiling; pcr; Bioinformatics
The link between ERG rearrangement and PTEN deletion is unclear in prostate cancer progression. Using fluorescence in situ hybridization, we systematically validated the frequency and distribution of ERG and PTEN aberrations in a cohort of 73 benign prostate tissues, 59 high grade prostatic intraepithelial neoplasia (HGPIN) foci, 281 localized prostate cancer and 47 androgen-independent metastatic prostate cancer patients. Overall, ERG rearrangement was present in 15% (5/33) of HGPIN, 45% (121/267) of localized cancers and 35% (15/43) of metastases. By contrast, PTEN deletion was identified in 9% (3/33) of HGPIN, 17% (42/251) of localized cancers and 54% (22/41) of metastases, of which 0%, 40% (17/42) and 45% (10/22) were homozygous, respectively. Concomitance of ERG rearrangement and PTEN deletion was observed in a subset of HGPIN. Significantly, association between PTEN deletion and ERG rearrangement was present both in localized cancers (p=0.0008) and metastases (p=0.02). Further, immunohistochemistry revealed significant correlation of decreased PTEN protein expression with PTEN genomic deletion both in localized and metastatic cancer. Of note, ERG aberration, but not PTEN deletion, was consistently identical both in localized cancer and adjacent HGPIN. Similarly, whereas all metastases (41/41, 100%) shared the same ERG status across multiple sites from the same patient, 5% (2/41) of cases showed discordance for PTEN deletion status across multiple sites. Collectively, our data support PTEN deletion as a late genetic event in human prostate cancer, presumably a “second hit” after ERG rearrangement. PTEN deletion and ERG rearrangement may cooperate, but contribute at different stages, in prostate cancer progression.
PTEN; Deletion; ERG; Rearrangement; Prostate cancer; Fluorescence in situ hybridization
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.
gene fusion; prostate cancer; ETS; DDX5; ETV4; fusion protein
Multiple, complex molecular events characterize cancer development and progression1,2. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumors, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high throughput liquid and gas chromatography-based mass spectrometry, we profiled more than 1126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer, and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly elevated during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also elevated in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase (GNMT), the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase (SARDH), induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Taken together, we profiled the metabolomic alterations of prostate cancer progression revealing sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.
Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and that regulates the survival and metastasis of cancer cells. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. Here, we show that the expression and function of EZH2 in cancer cell lines is inhibited by microRNA-101 (miR-101). Analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EZH2 expression. One or both of the two genomic loci encoding miR-101 were somatically lost in 37.5% of clinically localized prostate cancers (6/16) and 66.7% of metastatic disease (22/33). We propose that genomic loss of miR-101 in cancer leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.