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ABSTRACT: 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.
Current drug targets 02/2013; · 3.93 Impact Factor
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Irfan A Asangani,
Bushra Ateeq,
Qi Cao,
Lois Dodson,
Mithil Pandhi,
Lakshmi P Kunju,
Rohit Mehra,
Robert J Lonigro,
Javed Siddiqui,
Nallasivam Palanisamy,
Yi-Mi Wu,
Xuhong Cao,
Jung H Kim,
Meng Zhao,
Zhaohui S Qin,
Mathew K Iyer, Christopher A Maher,
Chandan Kumar-Sinha,
Sooryanarayana Varambally,
Arul M Chinnaiyan
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ABSTRACT: 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.
Molecular cell 11/2012; · 14.61 Impact Factor
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Dan R Robinson,
Shanker Kalyana-Sundaram,
Yi-Mi Wu,
Sunita Shankar,
Xuhong Cao,
Bushra Ateeq,
Irfan A Asangani,
Matthew Iyer, Christopher A Maher,
Catherine S Grasso, [......],
Xiaojun Jing,
Thomas J Giordano,
Michael S Sabel,
Celina G Kleer,
Nallasivam Palanisamy,
Rachael Natrajan,
Maryou B Lambros,
Jorge S Reis-Filho,
Chandan Kumar-Sinha,
Arul M Chinnaiyan
[show abstract]
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ABSTRACT: Breast cancer is a heterogeneous disease that has a wide range of molecular aberrations and clinical outcomes. Here we used 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 have a variety of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving genes encoding microtubule-associated serine-threonine kinase (MAST) and members of the Notch family. Both MAST and Notch-family gene fusions have substantial phenotypic effects in breast epithelial cells. Breast cancer cell lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and overexpression of MAST1 or MAST2 gene fusions has a proliferative effect both in vitro and in vivo. These findings show that recurrent gene rearrangements have key roles in subsets of carcinomas and suggest that transcriptome sequencing could identify individuals with rare, targetable gene fusions.
Nature medicine 11/2011; 17(12):1646-51. · 27.14 Impact Factor
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ABSTRACT: Prostate cancer is a common heterogeneous disease, and most patients diagnosed in the post prostate-specific antigen (PSA) era present with clinically localized disease, the majority of which do well regardless of treatment regimen undertaken. Overall, those with advanced prostate cancer at time of diagnosis do poorly after androgen withdrawal therapy. Understanding the biologic underpinning of prostate cancer is necessary to best determine the risk of disease progression and would be advantageous for the development of novel therapeutic approaches to impede or prevent disease. This review focuses on the recently identified common ETS and non-ETS gene rearrangements in prostate cancer. Although multiple molecular alterations have been detected in prostate cancer, a detailed understanding of gene fusion prostate cancer should help explain the clinical and biologic diversity, providing a rationale for a molecular subclassification of the disease.
Journal of Clinical Oncology 08/2011; 29(27):3659-68. · 18.37 Impact Factor
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Qi Cao,
Ram-Shankar Mani,
Bushra Ateeq,
Saravana M Dhanasekaran,
Irfan A Asangani,
John R Prensner,
Jung H Kim,
J Chad Brenner,
Xiaojun Jing,
Xuhong Cao, [......],
Mithil Pandhi,
Robert J Lonigro,
Yi-Mi Wu,
Scott A Tomlins,
Nallasivam Palanisamy,
Zhaohui Qin,
Jindan Yu, Christopher A Maher,
Sooryanarayana Varambally,
Arul M Chinnaiyan
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ABSTRACT: 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 upregulated 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 have uncovered a coordinate regulation of PRC1 and PRC2 activities that is mediated by miRNAs.
Cancer cell 08/2011; 20(2):187-99. · 25.29 Impact Factor
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ABSTRACT: 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.
http://chimerascan.googlecode.com
cmaher@dom.wustl.edu
Supplementary data are available at Bioinformatics online.
Bioinformatics 08/2011; 27(20):2903-4. · 5.47 Impact Factor
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Jung H Kim,
Saravana M Dhanasekaran,
John R Prensner,
Xuhong Cao,
Daniel Robinson,
Shanker Kalyana-Sundaram,
Christina Huang,
Sunita Shankar,
Xiaojun Jing,
Matthew Iyer, [......],
Lee Sam,
Catherine Grasso, Christopher A Maher,
Nallasivam Palanisamy,
Rohit Mehra,
Hal D Kominsky,
Javed Siddiqui,
Jindan Yu,
Zhaohui S Qin,
Arul M Chinnaiyan
[show abstract]
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ABSTRACT: Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression. We mapped the global DNA methylation patterns in select prostate tissues and cell lines using MethylPlex-next-generation sequencing (M-NGS). Hidden Markov model-based next-generation sequence analysis identified ∼68,000 methylated regions per sample. While global CpG island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation significantly increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively (P-value < 2 × 10(-16)). We found distinct patterns of promoter methylation around transcription start sites, where methylation occurred not only on the CGIs, but also on flanking regions and CGI sparse promoters. Among the 6691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) are cancer-specific, including numerous novel DMRs. A novel cancer-specific DMR in the WFDC2 promoter showed frequent methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization, and these modifications segregated into distinct regions when present on the same promoter. Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion-positive and -negative cancers, and we confirmed this observation using pyrosequencing on a tissue panel. This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.
Genome Research 07/2011; 21(7):1028-41. · 13.61 Impact Factor
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Yaping Sun,
Sooryanarayana Varambally, Christopher A Maher,
Qi Cao,
Peter Chockley,
Tomomi Toubai,
Chelsea Malter,
Evelyn Nieves,
Isao Tawara,
Yongqing Wang,
Peter A Ward,
Arul Chinnaiyan,
Pavan Reddy
[show abstract]
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ABSTRACT: While miRNAs are increasingly linked to various immune responses, whether they can be targeted for regulating in vivo inflammatory processes such as endotoxin-induced Gram-negative sepsis is not known. Production of cytokines by the dendritic cells (DCs) plays a critical role in response to endotoxin, lipopolysaccharide (LPS). We profiled the miRNA and mRNA of CD11c⁺ DCs in an unbiased manner and found that at baseline, miR-142-3p was among the most highly expressed endogenous miRs while IL-6 was among the most highly expressed mRNA after LPS stimulation. Multiple computational algorithms predicted the IL-6 3' untranslated region (UTR) to be a target of miR-142-3p. Studies using luciferase reporters carrying wild-type (WT) and mutant IL-6 3'UTR confirmed IL-6 as a target for miR-142-3p. In vitro knockdown and overexpression studies demonstrated a critical and specific role for miR142-3p in regulating IL-6 production by the DCs after LPS stimulation. Importantly, treatment of only WT but not the IL-6-deficient (IL-6(⁻/⁻)) mice with locked nucleic acid (LNA)-modified phosphorothioate oligonucleotide complementary to miR 142-3p reduced endotoxin-induced mortality. These results demonstrate a critical role for miR-142-3p in regulating DC responses to LPS and provide proof of concept for targeting miRs as a novel strategy for treatment of endotoxin-induced mortality.
Blood 06/2011; 117(23):6172-83. · 9.90 Impact Factor
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J Chad Brenner,
Bushra Ateeq,
Yong Li,
Anastasia K Yocum,
Qi Cao,
Irfan A Asangani,
Sonam Patel,
Xiaoju Wang,
Hallie Liang,
Jindan Yu, [......],
Jun Yang,
Scott A Tomlins, Christopher A Maher,
Kojo S J Elenitoba-Johnson,
Maha Hussain,
Nora M Navone,
Kenneth J Pienta,
Sooryanarayana Varambally,
Felix Y Feng,
Arul M Chinnaiyan
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ABSTRACT: 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.
Cancer cell 05/2011; 19(5):664-78. · 25.29 Impact Factor
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ABSTRACT: The second wave of next generation sequencing technologies, referred to as single-molecule sequencing (SMS), carries the promise of profiling samples directly without employing polymerase chain reaction steps used by amplification-based sequencing (AS) methods. To examine the merits of both technologies, we examine mRNA sequencing results from single-molecule and amplification-based sequencing in a set of human cancer cell lines and tissues. We observe a characteristic coverage bias towards high abundance transcripts in amplification-based sequencing. A larger fraction of AS reads cover highly expressed genes, such as those associated with translational processes and housekeeping genes, resulting in relatively lower coverage of genes at low and mid-level abundance. In contrast, the coverage of high abundance transcripts plateaus off using SMS. Consequently, SMS is able to sequence lower- abundance transcripts more thoroughly, including some that are undetected by AS methods; however, these include many more mapping artifacts. A better understanding of the technical and analytical factors introducing platform specific biases in high throughput transcriptome sequencing applications will be critical in cross platform meta-analytic studies.
PLoS ONE 01/2011; 6(3):e17305. · 4.09 Impact Factor
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John R Prensner,
Matthew K Iyer,
O Alejandro Balbin,
Saravana M Dhanasekaran,
Qi Cao,
J Chad Brenner,
Bharathi Laxman,
Irfan A Asangani,
Catherine S Grasso,
Hal D Kominsky,
Xuhong Cao,
Xiaojun Jing,
Xiaoju Wang,
Javed Siddiqui,
John T Wei,
Daniel Robinson,
Hari K Iyer,
Nallasivam Palanisamy, Christopher A Maher,
Arul M Chinnaiyan
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ABSTRACT: Noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease and to reveal uncharacterized aspects of tumor biology. Here we discover 121 unannotated prostate cancer-associated ncRNA transcripts (PCATs) by ab initio assembly of high-throughput sequencing of polyA(+) RNA (RNA-Seq) from a cohort of 102 prostate tissues and cells lines. We characterized one ncRNA, PCAT-1, as a prostate-specific regulator of cell proliferation and show that it is a target of the Polycomb Repressive Complex 2 (PRC2). We further found that patterns of PCAT-1 and PRC2 expression stratified patient tissues into molecular subtypes distinguished by expression signatures of PCAT-1-repressed target genes. Taken together, our findings suggest that PCAT-1 is a transcriptional repressor implicated in a subset of prostate cancer patients. These findings establish the utility of RNA-Seq to identify disease-associated ncRNAs that may improve the stratification of cancer subtypes.
Nature Biotechnology 01/2011; 29(8):742-9. · 29.50 Impact Factor
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Nallasivam Palanisamy,
Bushra Ateeq,
Shanker Kalyana-Sundaram,
Dorothee Pflueger,
Kalpana Ramnarayanan,
Sunita Shankar,
Bo Han,
Qi Cao,
Xuhong Cao,
Khalid Suleman, [......],
Douglas R Fullen,
Timothy M Johnson,
Joel K Greenson,
Thomas J Giordano,
Patrick Tan,
Scott A Tomlins,
Sooryanarayana Varambally,
Mark A Rubin, Christopher A Maher,
Arul M Chinnaiyan
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ABSTRACT: Although recurrent gene fusions involving erythroblastosis virus E26 transformation-specific (ETS) family transcription factors are common in prostate cancer, their products are considered 'undruggable' by conventional approaches. Recently, rare targetable gene fusions involving the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, have been identified in 1-5% of lung cancers, suggesting that similar rare gene fusions may occur in other common epithelial cancers, including prostate cancer. Here we used paired-end transcriptome sequencing to screen ETS rearrangement-negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF (solute carrier family 45, member 3-v-raf murine sarcoma viral oncogene homolog B1) and ESRP1-RAF1 (epithelial splicing regulatory protein-1-v-raf-1 murine leukemia viral oncogene homolog-1) gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and mitogen-activated protein kinase kinase (MAP2K1) inhibitors. Screening a large cohort of patients, we found that, although rare, recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers and melanoma. Taken together, our results emphasize the key role of RAF family gene rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion-harboring solid tumors and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.
Nature medicine 07/2010; 16(7):793-8. · 27.14 Impact Factor
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ABSTRACT: Protein-DNA interaction constitutes a basic mechanism for the genetic regulation of target gene expression. Deciphering this mechanism has been a daunting task due to the difficulty in characterizing protein-bound DNA on a large scale. A powerful technique has recently emerged that couples chromatin immunoprecipitation (ChIP) with next-generation sequencing, (ChIP-Seq). This technique provides a direct survey of the cistrom of transcription factors and other chromatin-associated proteins. In order to realize the full potential of this technique, increasingly sophisticated statistical algorithms have been developed to analyze the massive amount of data generated by this method.
Here we introduce HPeak, a Hidden Markov model (HMM)-based Peak-finding algorithm for analyzing ChIP-Seq data to identify protein-interacting genomic regions. In contrast to the majority of available ChIP-Seq analysis software packages, HPeak is a model-based approach allowing for rigorous statistical inference. This approach enables HPeak to accurately infer genomic regions enriched with sequence reads by assuming realistic probability distributions, in conjunction with a novel weighting scheme on the sequencing read coverage.
Using biologically relevant data collections, we found that HPeak showed a higher prevalence of the expected transcription factor binding motifs in ChIP-enriched sequences relative to the control sequences when compared to other currently available ChIP-Seq analysis approaches. Additionally, in comparison to the ChIP-chip assay, ChIP-Seq provides higher resolution along with improved sensitivity and specificity of binding site detection. Additional file and the HPeak program are freely available at http://www.sph.umich.edu/csg/qin/HPeak.
BMC Bioinformatics 01/2010; 11:369. · 2.75 Impact Factor
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Christopher A Maher,
Nallasivam Palanisamy,
John C Brenner,
Xuhong Cao,
Shanker Kalyana-Sundaram,
Shujun Luo,
Irina Khrebtukova,
Terrence R Barrette,
Catherine Grasso,
Jindan Yu,
Robert J Lonigro,
Gary Schroth,
Chandan Kumar-Sinha,
Arul M Chinnaiyan
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ABSTRACT: Recurrent gene fusions are a prevalent class of mutations arising from the juxtaposition of 2 distinct regions, which can generate novel functional transcripts that could serve as valuable therapeutic targets in cancer. Therefore, we aim to establish a sensitive, high-throughput methodology to comprehensively catalog functional gene fusions in cancer by evaluating a paired-end transcriptome sequencing strategy. Not only did a paired-end approach provide a greater dynamic range in comparison with single read based approaches, but it clearly distinguished the high-level "driving" gene fusions, such as BCR-ABL1 and TMPRSS2-ERG, from potential lower level "passenger" gene fusions. Also, the comprehensiveness of a paired-end approach enabled the discovery of 12 previously undescribed gene fusions in 4 commonly used cell lines that eluded previous approaches. Using the paired-end transcriptome sequencing approach, we observed read-through mRNA chimeras, tissue-type restricted chimeras, converging transcripts, diverging transcripts, and overlapping mRNA transcripts. Last, we successfully used paired-end transcriptome sequencing to detect previously undescribed ETS gene fusions in prostate tumors. Together, this study establishes a highly specific and sensitive approach for accurately and comprehensively cataloguing chimeras within a sample using paired-end transcriptome sequencing.
Proceedings of the National Academy of Sciences 08/2009; 106(30):12353-8. · 9.68 Impact Factor
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ABSTRACT: We have developed a tool, called ProbeMatch, for matching a large set of oligonucleotide sequences against a genome database using gapped alignments. Unlike most of the existing tools such as ELAND which only perform ungapped alignments allowing at most two mismatches, ProbeMatch generates both ungapped and gapped alignments allowing up to three errors including insertion, deletion and mismatch. To speedup sequence alignment, ProbeMatch uses gapped q-grams and q-grams of various patterns to identify target hits to a query sequence. This approach results in fewer initial sequences to examine with no loss in sensitivity. ProbeMatch has been used to align 169,095 Illumina GAII reads against the human genome, which could not be mapped by ELAND, and found alignments for 28,625 reads of the 169,095 reads in less than 3 h. AVAILABILITY: Source code is freely available at (http://www.cs.wisc.edu/~jignesh/probematch/).
Bioinformatics 05/2009; 25(11):1424-5. · 5.47 Impact Factor
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ABSTRACT: Recurrent gene fusions, typically associated with haematological malignancies and rare bone and soft-tissue tumours, have recently been described in common solid tumours. Here we use an integrative analysis of high-throughput long- and short-read transcriptome sequencing of cancer cells to discover novel gene fusions. As a proof of concept, we successfully used integrative transcriptome sequencing to 're-discover' the BCR-ABL1 (ref. 10) gene fusion in a chronic myelogenous leukaemia cell line and the TMPRSS2-ERG gene fusion in a prostate cancer cell line and tissues. Additionally, we nominated, and experimentally validated, novel gene fusions resulting in chimaeric transcripts in cancer cell lines and tumours. Taken together, this study establishes a robust pipeline for the discovery of novel gene chimaeras using high-throughput sequencing, opening up an important class of cancer-related mutations for comprehensive characterization.
Nature 02/2009; 458(7234):97-101. · 36.28 Impact Factor
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Sooryanarayana Varambally,
Qi Cao,
Ram-Shankar Mani,
Sunita Shankar,
Xiaosong Wang,
Bushra Ateeq,
Bharathi Laxman,
Xuhong Cao,
Xiaojun Jing,
Kalpana Ramnarayanan,
J Chad Brenner,
Jindan Yu,
Jung H Kim,
Bo Han,
Patrick Tan,
Chandan Kumar-Sinha,
Robert J Lonigro,
Nallasivam Palanisamy, Christopher A Maher,
Arul M Chinnaiyan
[show abstract]
[hide abstract]
ABSTRACT: Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and 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 are 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 cancer cells (6 of 16) and 66.7% of metastatic disease cells (22 of 33). We propose that the genomic loss of miR-101 in cancer leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.
Science 12/2008; 322(5908):1695-9. · 31.20 Impact Factor
