-
[show abstract]
[hide abstract]
ABSTRACT: RNA processing is vital for the high fidelity and diversity of eukaryotic transcriptomes and the encoded proteomes. However, control of RNA processing is not fully established. Σ RNA is a class of conserved large non-coding RNAs (murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense technology, we identified that RNA post-transcriptional modification is the most significant global function of Σ RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of Σ RNA/MALAT-1. These results support the hypothesis that Σ RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification.
FEBS letters 02/2011; 585(4):671-6. · 3.54 Impact Factor
-
Eran Elyakim,
Einat Sitbon,
Alexander Faerman,
Sarit Tabak,
Eve Montia,
Liron Belanis,
Avital Dov, Eric G Marcusson,
C Frank Bennett,
Ayelet Chajut,
Dalia Cohen,
Noga Yerushalmi
[show abstract]
[hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.
Cancer Research 10/2010; 70(20):8077-87. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The tropism of breast cancer cells for bone and their tendency to induce an osteolytic phenotype are a result of interactions between breast cancer cells and stromal cells and are of paramount importance for bone metastasis. However, the underlying molecular mechanisms remain poorly understood. We hypothesize that tumor-stromal interaction alters gene expression in malignant tumor cells and stromal cells creating a unique expression signature that promotes osteolytic breast cancer bone metastasis and that inhibition of such interactions can be developed as targeted therapeutics. Microarray analysis was performed to investigate gene expression profiling at the tumor-bone (TB) interface versus the tumor alone area from syngenic mice injected with three different syngenic mammary tumor cell lines that differ in their metastatic potential. We identified matrix metalloproteinase 13 (MMP13), receptor activator of NF-kappaB ligand (RANKL), and integrins binding sialoprotein to be genes upregulated at the TB interface and validated. To determine the functional role of MMP13 in tumor-induced osteolysis, mice with Cl66 mammary tumors were treated with MMP13 antisense oligonucleotides (MMP13-ASO) or control scrambled oligonucleotides (control-ASO). Knockdown of MMP13 expression at the TB interface leads to significant reduction in bone destruction and in the number of activated osteoclasts at the TB interface. Further analysis to evaluate the mechanism of MMP13-dependent osteolytic bone metastasis revealed that MMP13-ASO treatment decreased active MMP9, RANKL levels, and transforming growth factor-beta signaling at the TB interface. Together, our data indicate that upregulation of MMP13 at the TB interface is important in tumor-induced osteolysis and suggest that MMP13 is a potential therapeutic target for breast cancer bone metastasis.
Cancer Research 05/2010; 70(9):3494-504. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are increasingly implicated in the regulation of metastasis. Despite their potential as targets for anti-metastatic therapy, miRNAs have only been silenced in normal tissues of rodents and nonhuman primates. Therefore, the development of effective approaches for sequence-specific inhibition of miRNAs in tumors remains a scientific and clinical challenge. Here we show that systemic treatment of tumor-bearing mice with miR-10b antagomirs-a class of chemically modified anti-miRNA oligonucleotide-suppresses breast cancer metastasis. Both in vitro and in vivo, silencing of miR-10b with antagomirs significantly decreases miR-10b levels and increases the levels of a functionally important miR-10b target, Hoxd10. Administration of miR-10b antagomirs to mice bearing highly metastatic cells does not reduce primary mammary tumor growth but markedly suppresses formation of lung metastases in a sequence-specific manner. The miR-10b antagomir, which is well tolerated by normal animals, appears to be a promising candidate for the development of new anti-metastasis agents.
Nature Biotechnology 03/2010; 28(4):341-7. · 29.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers in relation to disease progression and drives cellular transformation, tumorigenesis, and metastatic progression in experimental models. Enhanced eIF4E function results from eIF4E overexpression and/or activation of the ras and phosphatidylinositol 3-kinase/AKT pathways and selectively increases the translation of key mRNAs involved in tumor growth, angiogenesis, and cell survival. Consequently, by simultaneously and selectively reducing the expression of numerous potent growth and survival factors critical for malignancy, targeting eIF4E for inhibition may provide an attractive therapy for many different tumor types. Recent work has now shown the plausibility of therapeutically targeting eIF4E and has resulted in the advance of the first eIF4E-specific therapy to clinical trials. These studies illustrate the increased susceptibility of tumor tissues to eIF4E inhibition and support the notion that the enhanced eIF4E function common to many tumor types may represent an Achilles' heel for cancer.
Cancer Research 03/2008; 68(3):631-4. · 7.86 Impact Factor
-
Jeremy R Graff,
Bruce W Konicek,
Thomas M Vincent,
Rebecca L Lynch,
David Monteith,
Spring N Weir,
Phil Schwier,
Andrew Capen,
Robin L Goode,
Michele S Dowless, [......],
Lillian Sams,
Sandaruwan Geeganage,
Larry E Douglass,
Blake Lee Neubauer,
Nicholas M Dean,
Kerry Blanchard,
Jianyong Shou,
Louis F Stancato,
Julia H Carter, Eric G Marcusson
[show abstract]
[hide abstract]
ABSTRACT: Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
Journal of Clinical Investigation 10/2007; 117(9):2638-48. · 15.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC.
Cancer Research 05/2007; 67(8):3583-93. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Adipogenesis is the process by which undifferentiated precursor cells differentiate into fat laden adipocytes. The nuclear proteins peroxisome proliferator-activated receptors (PPARs) play a central role in adipocyte differentiation. The goals of this study were to identify novel PPARgamma responsive genes and to determine their role in regulating human adipocyte differentiation. Affymetrix profiling of gene expression in human adipocytes identified about 1000 genes that were significantly up-regulated subsequent to induction of differentiation. PPARgamma expression was reduced prior to induction of differentiation using a novel, chemically modified antisense oligonucleotide. Affymetrix microarray profiling of these cells identified 278 statistically significantly down-regulated genes. Eight genes were found to contain previously documented PPARgamma recognition element (PPRE) in their upstream nucleotide (promoter) sequence. Four of these genes are novel and have not previously been characterized. Chromatin immuno-precipitation experiments confirmed the binding of PPARgamma to the PPRE of three of these genes. The ortholog of one of these genes, hypothetical protein FLJ 20920, has previously been reported to be involved in the control of body fat composition in Caenorhabditis elegans. Inhibition of expression of this protein was found to also inhibit differentiation of human adipocytes. MAST/MEME algorithm analysis was used to identify novel commonly occurring sequence motifs in the 5' upstream region of transcripts for subset of down-regulated genes, which were grouped according to their sequence similarities. A number of clusters were identified and the largest cluster contained similar motifs from 26 genes with the literature supporting 7 of the 26 genes as being involved in fatty acid metabolism or PPARgamma interaction.
Gene 04/2006; 369:90-9. · 2.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.
Nucleic Acids Research 02/2006; 34(16):4467-76. · 8.03 Impact Factor
-
Christine Esau,
Xiaolin Kang,
Eigen Peralta,
Elaine Hanson, Eric G Marcusson,
Lingamanaidu V Ravichandran,
Yingqing Sun,
Seongjoon Koo,
Ranjan J Perera,
Ravi Jain,
Nicholas M Dean,
Susan M Freier,
C Frank Bennett,
Bridget Lollo,
Richard Griffey
[show abstract]
[hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.
Journal of Biological Chemistry 01/2005; 279(50):52361-5. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The application of antisense technology to study physiological and disease processes continues to mature. Antisense approaches are among the most direct means to use genomic sequence information. When developing therapeutics, applications range from early target validation in discovery to the therapeutic product. In this review, we describe the application of antisense oligonucleotides (ASOs) to identify genes that are important in controlling angiogenesis. High-throughput assays in vitro have been used to evaluate many gene targets. Genes that appear to be important in angiogenesis are then evaluated further in animal models of ocular angiogenesis. The ability of ASOs to reduce target-gene expression in the appropriate cells in the eye raises the possibility that this class of compounds could be used for target validation in vivo, and also be developed as a novel class of therapeutics in their own right.
Trends in Pharmacological Sciences 11/2004; 25(10):523-7. · 10.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases. The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions. Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets. This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets. The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models. Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs. The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.
Oligonucleotides 02/2004; 14(1):49-64. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Adipogenesis is the process by which undifferentiated precursor cells differentiate into fat laden adipocytes. The nuclear proteins peroxisome proliferator-activated receptors (PPARs) play a central role in adipocyte differentiation. The goals of this study were to identify novel PPARγ responsive genes and to determine their role in regulating human adipocyte differentiation. Affymetrix profiling of gene expression in human adipocytes identified about 1000 genes that were significantly up-regulated subsequent to induction of differentiation. PPARγ expression was reduced prior to induction of differentiation using a novel, chemically modified antisense oligonucleotide. Affymetrix microarray profiling of these cells identified 278 statistically significantly down-regulated genes. Eight genes were found to contain previously documented PPARγ recognition element (PPRE) in their upstream nucleotide (promoter) sequence. Four of these genes are novel and have not previously been characterized. Chromatin immuno-precipitation experiments confirmed the binding of PPARγ to the PPRE of three of these genes. The ortholog of one of these genes, hypothetical protein FLJ 20920, has previously been reported to be involved in the control of body fat composition in Caenorhabditis elegans. Inhibition of expression of this protein was found to also inhibit differentiation of human adipocytes. MAST/MEME algorithm analysis was used to identify novel commonly occurring sequence motifs in the 5′ upstream region of transcripts for subset of down-regulated genes, which were grouped according to their sequence similarities. A number of clusters were identified and the largest cluster contained similar motifs from 26 genes with the literature supporting 7 of the 26 genes as being involved in fatty acid metabolism or PPARγ interaction.
Gene.
