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Amjad P Khan,
Thekkelnaycke M Rajendiran,
Bushra Ateeq, Irfan A Asangani,
Jyoti N Athanikar,
Anastasia K Yocum,
Rohit Mehra,
Javed Siddiqui,
Ganesh Palapattu,
John T Wei,
George Michailidis,
Arun Sreekumar,
Arul M Chinnaiyan
[show abstract]
[hide abstract]
ABSTRACT: Metabolomic profiling of prostate cancer (PCa) progression identified markedly elevated levels of sarcosine (N-methyl glycine) in metastatic PCa and modest but significant elevation of the metabolite in PCa urine. Here, we examine the role of key enzymes associated with sarcosine metabolism in PCa progression. Consistent with our earlier report, sarcosine levels were significantly elevated in PCa urine sediments compared to controls, with a modest area under the receiver operating characteristic curve of 0.71. In addition, the expression of sarcosine biosynthetic enzyme, glycine N-methyltransferase (GNMT), was elevated in PCa tissues, while sarcosine dehydrogenase (SARDH) and pipecolic acid oxidase (PIPOX), which metabolize sarcosine, were reduced in prostate tumors. Consistent with this, GNMT promoted the oncogenic potential of prostate cells by facilitating sarcosine production, while SARDH and PIPOX reduced the oncogenic potential of prostate cells by metabolizing sarcosine. Accordingly, addition of sarcosine, but not glycine or alanine, induced invasion and intravasation in an in vivo PCa model. In contrast, GNMT knockdown or SARDH overexpression in PCa xenografts inhibited tumor growth. Taken together, these studies substantiate the role of sarcosine in PCa progression.
Neoplasia (New York, N.Y.) 05/2013; 15(5):491-501. · 5.48 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
[show abstract]
[hide abstract]
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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Rui Wang, Irfan A Asangani,
Balabhadrapatruni V S K Chakravarthi,
Bushra Ateeq,
Robert J Lonigro,
Qi Cao,
Ram-Shankar Mani,
Daniel F Camacho,
Natalie Mcgregor,
Taibriana E W Schumann, [......],
Rohit Mehra,
Javed Siddiqui,
Saravana M Dhanasekaran,
Mukesh K Nyati,
Kenneth J Pienta,
Nallasivam Palanisamy,
Lakshmi P Kunju,
Mark A Rubin,
Arul M Chinnaiyan,
Sooryanarayana Varambally
[show abstract]
[hide abstract]
ABSTRACT: 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 sup-pressors 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 signifi-cance 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
Neoplasia 10/2012; · 5.95 Impact Factor
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Rui Wang, Irfan A Asangani,
Balabhadrapatruni Vsk Chakravarthi,
Bushra Ateeq,
Robert J Lonigro,
Qi Cao,
Ram-Shankar Mani,
Daniel F Camacho,
Natalie McGregor,
Taibriana Ew Schumann, [......],
Rohit Mehra,
Javed Siddiqui,
Saravana M Dhanasekaran,
Mukesh K Nyati,
Kenneth J Pienta,
Nallasivam Palanisamy,
Lakshmi P Kunju,
Mark A Rubin,
Arul M Chinnaiyan,
Sooryanarayana Varambally
[show abstract]
[hide abstract]
ABSTRACT: 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.
Neoplasia (New York, N.Y.) 10/2012; 14(10):905-14. · 5.48 Impact Factor
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Irfan A Asangani,
Paul W Harms,
Lois Dodson,
Mithil Pandhi,
Lakshmi P Kunju,
Chris A Maher,
Douglas R Fullen,
Timothy M Johnson,
Thomas J Giordano,
Nallasivam Palanisamy,
Arul M Chinnaiyan
[show abstract]
[hide abstract]
ABSTRACT: MicroRNAs (miRs) play a key role in cancer etiology by coordinately repressing numerous target genes involved in cell proliferation, migration and invasion. The genomic region in chromosome 9p21 that encompasses miR-31 is frequently deleted in solid cancers including melanoma; however the expression and functional role of miR-31 has not been previously studied in melanoma. Here, we queried the expression status and performed functional characterization of miR-31 in melanoma tissues and cell lines. We found that down-regulation of miR-31 was a common event in melanoma tumors and cell lines and was associated with genomic loss in a subset of samples. Down-regulation of miR-31 gene expression was also a result of epigenetic silencing by DNA methylation, and via EZH2-mediated histone methylation. Ectopic overexpression of miR-31 in various melanoma cell lines inhibited cell migration and invasion. miR-31 targets include oncogenic kinases such as SRC, MET, NIK (MAP3K14) and the melanoma specific oncogene RAB27a. Furthermore, miR-31 overexpression resulted in down-regulation of EZH2 and a de-repression of its target gene rap1GAP; increased expression of EZH2 was associated with melanoma progression and overall patient survival. Taken together, our study supports a tumor suppressor role for miR-31 in melanoma and identifies novel therapeutic targets.
Oncotarget 08/2012; · 4.78 Impact Factor
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Shanker Kalyana-Sundaram,
Chandan Kumar-Sinha,
Sunita Shankar,
Dan R Robinson,
Yi-Mi Wu,
Xuhong Cao, Irfan A Asangani,
Vishal Kothari,
John R Prensner,
Robert J Lonigro,
Matthew K Iyer,
Terrence Barrette,
Achiraman Shanmugam,
Saravana M Dhanasekaran,
Nallasivam Palanisamy,
Arul M Chinnaiyan
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cell 06/2012; 149(7):1622-34. · 32.40 Impact Factor
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Catherine S Grasso,
Yi-Mi Wu,
Dan R Robinson,
Xuhong Cao,
Saravana M Dhanasekaran,
Amjad P Khan,
Michael J Quist,
Xiaojun Jing,
Robert J Lonigro,
J Chad Brenner, [......],
John R Prensner,
Nallasivam Palanisamy,
Gregory A Ryslik,
Fabio Vandin,
Benjamin J Raphael,
Lakshmi P Kunju,
Daniel R Rhodes,
Kenneth J Pienta,
Arul M Chinnaiyan,
Scott A Tomlins
[show abstract]
[hide abstract]
ABSTRACT: Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. 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 signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) 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 signalling deregulated in prostate cancer, and prioritize candidates for future study.
Nature 05/2012; 487(7406):239-43. · 36.28 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]
[hide abstract]
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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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
[show abstract]
[hide abstract]
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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Xiao-Song Wang,
Sunita Shankar,
Saravana M Dhanasekaran,
Bushra Ateeq,
Atsuo T Sasaki,
Xiaojun Jing,
Daniel Robinson,
Qi Cao,
John R Prensner,
Anastasia K Yocum, [......],
Gilbert S Omenn,
Dorothee Pflueger,
Anuradha Gopalan,
Victor E Reuter,
Emily Rose Kahoud,
Lewis C Cantley,
Mark A Rubin,
Nallasivam Palanisamy,
Sooryanarayana Varambally,
Arul M Chinnaiyan
[show abstract]
[hide abstract]
ABSTRACT: Using an integrative genomics approach called amplification breakpoint ranking and assembly analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, a specific knockdown of which attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 mitogen-activated protein kinase (MAPK) pathways. This is the first report of a gene fusion involving the Ras family, suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. SIGNIFICANCE: This is the first description of an oncogenic gene fusion of KRAS, one of the most studied proto-oncogenes. KRAS rearrangement may represent the driving mutation in a rare subset of metastatic prostate cancers, emphasizing the importance of RAS-RAF-MAPK signaling in this disease.
Cancer discovery. 06/2011; 1(1):35-43.
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[show abstract]
[hide abstract]
ABSTRACT: Curcumin has promising potential in cancer prevention and therapy by interacting with proteins and modifying their expression and activity, which includes transcription factors, inflammatory cytokines and factors of cell survival, proliferation and angiogenesis. miR-21 is overexpressed in many tumours, promoting progression and metastasis. In the present study, we examined the potential of curcumin to regulate miR-21, tumour growth, invasion and in vivo metastasis in colorectal cancer. In Rko and HCT116 cells, we identified two new transcriptional start sites of the miR-21 gene and delineated its promoter region. PMA stimulation induced miR-21 expression via motifs bound with AP-1 (activator protein 1) transcription factors. Curcumin treatment reduced miR-21 promoter activity and expression in a dose-dependent manner by inhibiting AP-1 binding to the promoter, and induced the expression of the tumour suppressor Pdcd4 (programmed cell death protein 4), which is a target of miR-21. Curcumin-treated Rko and HCT116 cells were arrested in the G2/M phase with increasing concentrations. Furthermore, curcumin inhibited tumour growth, invasion and in vivo metastasis in the chicken-embryo-metastasis assay [CAM (chorionallantoic membrane) assay]. Additionally, curcumin significantly inhibited miR-21 expression in primary tumours generated in vivo in the CAM assay by Rko and HCT116 cells (P<0.00006 and P<0.035 respectively). Taken together, this is the first paper to show that curcumin inhibits the transcriptional regulation of miR-21 via AP-1, suppresses cell proliferation, tumour growth, invasion and in vivo metastasis, and stabilizes the expression of the tumour suppressor Pdcd4 in colorectal cancer.
Bioscience Reports 06/2011; 31(3):185-97. · 2.38 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
[show abstract]
[hide abstract]
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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Bushra Ateeq,
Scott A Tomlins,
Bharathi Laxman, Irfan A Asangani,
Qi Cao,
Xuhong Cao,
Yong Li,
Xiaoju Wang,
Felix Y Feng,
Kenneth J Pienta,
Sooryanarayana Varambally,
Arul M Chinnaiyan
[show abstract]
[hide abstract]
ABSTRACT: Gene fusions involving ETS (erythroblastosis virus E26 transformation-specific) family transcription factors are found in ~50% of prostate cancers and as such can be used as a basis for the molecular subclassification of prostate cancer. Previously, we showed that marked overexpression 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. Recombinant SPINK1 protein (rSPINK1) stimulated cell proliferation in benign RWPE as well as cancerous prostate cells. Indeed, RWPE cells treated with either rSPINK1 or conditioned medium from 22RV1 prostate cancer cells (SPINK1+/ETS⁻) significantly increased cell invasion and intravasation when compared with untreated cells. In contrast, knockdown of SPINK1 in 22RV1 cells inhibited cell proliferation, cell invasion, and tumor growth in xenograft assays. 22RV1 cell proliferation, invasion, and intravasation were attenuated by a monoclonal antibody (mAb) to SPINK1 as well. We also demonstrated that SPINK1 partially mediated its neoplastic effects through interaction with the epidermal growth factor receptor (EGFR). Administration of antibodies to SPINK1 or EGFR (cetuximab) in mice bearing 22RV1 xenografts attenuated tumor growth by more than 60 and 40%, respectively, or ~75% when combined, without affecting PC3 xenograft (SPINK1⁻/ETS⁻) growth. Thus, this study suggests that SPINK1 may be a therapeutic target in a subset of patients with SPINK1+/ETS⁻ prostate cancer. Our results provide a rationale for both the development of humanized mAbs to SPINK1 and evaluation of EGFR inhibition in SPINK1+/ETS⁻ prostate cancers.
Science translational medicine 03/2011; 3(72):72ra17. · 7.80 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
[show abstract]
[hide abstract]
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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Dessislava A Nikolova, Irfan A Asangani,
Laura D Nelson,
Dennis P M Hughes,
Doris R Siwak,
Gordon B Mills,
Andrea Harms,
Erika Buchholz,
Lothar R Pilz,
Christian Manegold,
Heike Allgayer
[show abstract]
[hide abstract]
ABSTRACT: Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non-small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif -190/-171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab.
Cancer Research 04/2009; 69(6):2461-70. · 7.86 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Ubiquitously expressed micro- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region -187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.
Gene 03/2008; 410(1):197-206. · 2.34 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2-related factor to promoter region -152/-135 of the metastasis-related u-PAR gene in 60% of in vivo-resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (-190/-171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor-binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions.
In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region -190/-171 was done in tumors and normal tissues. In 71 patients, region -152/-135 was also analyzed. U-PAR protein was measured by ELISA.
Tumor-specific AP-1 binding to region -190/-171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P<0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P<0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P=0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors.
This is the first study differentiating transcription factor-binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.
Clinical Cancer Research 01/2006; 11(24 Pt 1):8538-48. · 7.74 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Ubiquitously expressed µ- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~ 2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region − 187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.
Gene.
