Long-term Silencing Research Articles

Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

Abstract PR03: Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for DNA methylation maintenance

Abstract DNA methylation and histone post-translational modifications (PTMs) represent two key epigenetic regulators of DNA information in chromatin. The faithful inheritance of DNA methylation is essential for normal mammalian development and long-term transcriptional silencing, and aberrant DNA methylation patterns are a hallmark of many cancers. Histone PTMs can alter the physical structure of chromatin and have been proposed to function in the context of a “histone code” to coordinate the recruitment of effector proteins that elicit selective effects on chromatin-templated cellular processes like gene transcription and DNA repair. Indeed, defects in histone recognition by effector proteins are also emerging as underlying causes of cancer initiation and progression. Effector proteins often contain multiple chromatin binding domains. While significant progress has been made characterizing individual effector domains, the role of paired domains and how they function in a combinatorial fashion within chromatin is poorly defined. The E3 ubiquitin ligase UHRF1 is a unique chromatin effector protein, in that it integrates the recognition of both histone PTMs and DNA methylation. We recently showed that H3K9 methyl binding by the tandem Tudor domain (TTD) of UHRF1 is required for DNA methylation maintenance in cancer cells — yet the function of its adjacent plant homeodomain (PHD) in this process was unknown. Here we show that the linked TTD and PHD of UHRF1 operate as a single functional unit in cells, providing a defined combinatorial readout of the modification state of the histone H3 tail that is essential for UHRF1-directed DNA methylation maintenance by DNMT1. These findings provide critical support for the ‘histone code' hypothesis, demonstrating that multivalent histone engagement plays a fundamental role in driving a downstream biological event in chromatin and suggest UHRF1 may be a favorable therapeutic target for regulating aberrant DNA methylation in cancers. This abstract is also presented as Poster B26. Citation Format: Scott B. Rothbart, Bradley M. Dickson, Michelle S. Ong, Krzysztof Krajewski, Scott Houliston, Dmitri B. Kireev, Cheryl H. Arrowsmith, Brian D. Strahl. Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for DNA methylation maintenance. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR03.

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition(1). Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce(2). Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration(3)Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study(4). Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Ataxin-3 protein modification as a treatment strategy for spinocerebellar ataxia type 3: Removal of the CAG containing exon

Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3.

Abstract 1513: MLH1 silencing is caused by hypoxia through epigenetic regulation..

Abstract Transcriptional silencing of the DNA mismatch repair (MMR) gene, MLH1, is one of the main causes of microsatellite instability in sporadic cancers of the colon and other sites. We and others have shown that hypoxia, a key microenvironmental stress in solid tumors, causes reduced expression of MLH1via transient transcriptional repression mediated by both HIF-dependent and independent pathways. Here, we further investigated whether hypoxia-induced down-regulation of MLH1 is associated with epigenetic modifications that may mark the locus for long-term silencing. We find that hypoxia drives epigenetic repression of the MLH1 promoter with decreased H3K4 methylation as a key repressive modification. We find that this is mediated by the H3K4 demethylases, LSD1 (lysine-specific histone demethylase 1) and PLU-1/JARID1B. In RKO cells containing a reporter construct consisting of the MLH1 promoter driving selectable thymidine kinase (TK) and blasticidin resistance (BlastR) genes, long-term silencing of the MLH1 promoter was produced by exposure of the cells to hypoxia as compared to minimal silencing under normoxia. Importantly, knockdown of LSD1 blocked the hypoxia-induced MLH1 promoter silencing in this reporter system. In RKO cells, the endogenous MLH1 promoter is constitutively silenced by DNA hypermethylation at CpG sites. MLH1 re-expression can be induced by 5-aza-2-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, accompanied by increased levels of H3K9 acetylation and H3K4 methylation at it promoter. But the promoter rapidly becomes re-silenced after removal of 5-Aza-dC. However, we find that knockdown of LSD1 or its corepressor, CoRest, blocks MLH1 resilencing in the RKO cells. Consistently, ChIP analyses revealed that the control RKO cells (with a GFP knockdown vector) showed return of both H3K9 acetylation and H3K4 methylation back to pre-treatment levels coupled with MLH1 re-silencing upon removal of 5-Aza-dC, but intriguingly, high levels of H3K9 acetylation and H3K4 methylation were also observed at MLH1 promoters in RKO LSD1sh cells. Analysis of DNA methylation revealed that MLH1 promoter methylation levels were reduced by 5-Aza-dC treatment, as expected. However, after removal of 5-Aza-dC, promoter methylation levels returned back to the pre-treatment levels only in the control RKO cells. In the RKO cells with shRNA mediated knock down of either LSD1 or CoRest, the promoter re-methylation process was blocked. Collectively, the results demonstrate that the LSD1/CoRest repressive complex is essential for the MLH1 silencing and that hypoxia is a critical driving force for stable transcriptional silencing of MLH1 through a concerted pattern of chromatin and DNA modifications, thereby promoting genetic instability and tumor progression. Citation Format: Yuhong Lu, Narendra Wajapeyee, Mitchell S. Turker, Peter M. Glazer. MLH1 silencing is caused by hypoxia through epigenetic regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1513. doi:10.1158/1538-7445.AM2013-1513

How to Analyze DNA Methylation?

DNA methylation involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring (cytosine and adenine are two of the four bases of DNA). DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation plays multiple roles during development and serves to establish long-term gene silencing. Mapping of DNA methylation changes especially in CpG islands has become essential for the understanding of diverse biological processes. There is an important relationship between the degree of DNA methylation and gene activity in cancer. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. By discovering sites of aberrant methylation in tumors, we can identify potential new biomarkers suitable for diagnostic and prognostic testing. A number of techniques for analysis of DNA methylation have been reported. The aim of this study was to review methodologies that can be used for monitoring of DNA methylation changes. We describe the advantages, disadvantages and potential use of these techniques. Key words: DNA methylation, bisulfite conversion, MSP, MS-MLPA, restriction endonucleases

MYCN is recruited to the RASSF1A promoter but is not critical for DNA hypermethylation in neuroblastoma

Tumor suppressor genes such as RASSF1A are often epigenetically repressed by DNA hypermethylation in neuroblastoma, where the MYCN proto-oncogene is frequently amplified. MYC has been shown to associate with DNA methyltransferases, thereby inducing transcriptional repression of target genes, which suggested that MYCN might play a similar mechanistic role in the hypermethylation of tumor suppressor genes in neuroblastoma. This study tested that hypothesis by using co-immunoprecipitation and ChIP to investigate MYCN-DNA methyltransferase interactions, together with MYCN knock-down and over-expression systems to examine the effect of MYCN expression changes on gene methylation, employing both candidate gene and genome-wide assays. We show that MYCN interacts with DNA methyltransferases and is recruited to the promoter region of RASSF1A. However, using four model systems, we showed that long-term silencing of MYCN induces only a small loss of DNA methylation at the RASSF1A promoter in MYCN amplified neuroblastoma cell lines and over-expression of MYCN does not induce any DNA methylation, suggesting that MYCN is not critical for DNA hypermethylation in neuroblastoma.

Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx.

Integrated retroviral DNA is subject to epigenetic transcriptional silencing at different frequencies. This process is mediated by repressive DNA methylation and histone modifications on viral chromatin. However, the detailed mechanisms by which retroviral silencing is initiated and maintained are not well understood. Using a model system in which avian sarcoma virus (ASV) DNA is epigenetically repressed in mammalian cells, we previously found that a cellular scaffolding protein, Daxx, acts as an antiretroviral factor that promotes epigenetic repression through recruitment of histone deacetylases (HDACs). Here we show that human Daxx protein levels are increased in response to retroviral infection and that Daxx acts at the time of infection to initiate epigenetic repression. Consistent with a rapid and active antiviral epigenetic response, we found that repressive histone marks and long terminal repeat (LTR) DNA methylation could be detected within 12 h to 3 days postinfection, respectively. Daxx was also found to be required for long-term ASV silencing maintenance and full viral DNA methylation, and it was physically associated with both viral DNA and DNA methyltransferases (DNMTs). These findings support a model in which incoming retroviral protein-DNA complexes are detected by Daxx, and the integrated provirus is rapidly chromatinized and repressed by DNA methylation and histone modification as part of an antiviral response. These results uncover a possible direct and active antiviral mechanism by which DNMTs can be recruited to retroviral DNA.

PiRNAs Can Trigger a Multigenerational Epigenetic Memory in the Germline of C. elegans

SummaryTransgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use.

Stilbene derivatives promote Ago2-dependent tumour-suppressive microRNA activity

It is well known that natural products are a rich source of compounds for applications in medicine, pharmacy, and biology. However, the exact molecular mechanisms of natural agents in human health have not been clearly defined. Here, we demonstrate for the first time that the polyphenolic phytoalexin resveratrol promotes expression and activity of Argonaute2 (Ago2), a central RNA interference (RNAi) component, which thereby inhibits breast cancer stem-like cell characteristics by increasing the expression of a number of tumour-suppressive miRNAs, including miR-16, -141, -143, and -200c. Most importantly, resveratrol-induced Ago2 resulted in a long-term gene silencing response. We also found that pterostilbene, which is a natural dimethylated resveratrol analogue, is capable of mediating Ago2-dependent anti-cancer activity in a manner mechanistically similar to that of resveratrol. These findings suggest that the dietary intake of natural products contributes to the prevention and treatment of diseases by regulating the RNAi pathway.

Abstract IA20: Targeting of microRNAs for therapeutics

Abstract microRNAs act as important post-transcriptional regulators of gene expression by mediating mRNA degradation and translational repression. Moreover, there is ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Besides cancer, miRNAs have also been implicated in viral infections, cardiovascular and muscle diseases and CNS disorders. Thus, disease-associated miRNAs represent a potential new class of targets for oligonucleotide-based therapeutics, which may yield patient benefits unobtainable by other therapeutic approaches. LNA is a bicyclic high-affinity RNA analogue, in which the ribose ring is locked in an RNA-like, N-type (C3′-endo) conformation by the introduction of a 2′-O,4′-C methylene bridge. Transfection of LNA-modified antimiR oligonucleotides into cells results in potent and specific inhibition of miRNA function with concomitant derepression of direct target mRNAs. In addition, systemically delivered, unconjugated LNA-antimiRs with a phosphorothioate backbone show high metabolic stability and uptake in many tissues in mice, coinciding with long-term miRNA silencing in vivo. These findings support the utility of LNA-modified antimiR oligonucleotides in the development of therapeutic strategies aimed at pharmacological inhibition of disease-associated miRNAs. We will describe recent progress in targeting of disease-implicated miRNAs for therapeutics using LNA-antimiRs and present an update on the clinical development of antimiR-122 for treatment of hepatitis C virus infection. Citation Format: Sakari Kauppinen. Targeting of microRNAs for therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA20.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

Stapled Peptides as Anti-Apoptotic Drugs

A major problem with many cancer chemotherapeutic drugs is their lack of selectivity. They attack and kill not only the cancer target of interest, but in many cases also normal cells in tissues that are vital for cancer patient survival. Drugs that target cellular functions that are essential specifically for cancer cell survival should thus be of benefit to the cancer patient undergoing chemotherapy.Cancer cells have adapted numerous strategies to avoid apoptosis, or programmed cell death. The BCL-2 family contains anti-apoptotic and pro-apoptotic proteins. The anti-apoptotic BCL-2 family proteins have been linked to the survival of pathogenic cells that include cancer cells. RNA inhibition has been one approach to blocking anti-apoptotic BCL-2 and hence cancer cell survival. However, there is a question about the effectiveness of siRNA in long-term gene silencing.

Chemically Modified Oligonucleotides Modulate an Epigenetically Varied and Transient Form of Transcription Silencing of HIV-1 in Human Cells

Small noncoding RNAs (ncRNAs) have been shown to guide epigenetic silencing complexes to target loci in human cells. When targeted to gene promoters, these small RNAs can lead to long-term stable epigenetic silencing of gene transcription. To date, small RNAs have been shown to modulate transcriptional gene silencing (TGS) of human immunodeficiency virus type 1 (HIV-1) as well as several other disease-related genes, but it has remained unknown as to what extent particular chemistries can be used to generate single-stranded backbone-modified oligonucleotides that are amenable to this form of gene targeting and regulation. Here, we present data indicating that specific combinations of backbone modifications can be used to generate single-stranded antisense oligonucleotides that can functionally direct TGS of HIV-1 in a manner that is however, independent of epigenetic changes at the target loci. Furthermore, this functionality appears contingent on the absence of a 5′ phosphate in the oligonucleotide. These data suggest that chemically modified oligonucleotide based approaches could be implemented as a means to regulate gene transcription in an epigenetically independent manner.

SiRNA delivery with chitosan nanoparticles: Molecular properties favoring efficient gene silencing

Chitosan has gained increasing interest for siRNA delivery. Although chitosan covers a family of structurally different polysaccharides, most siRNA delivery studies have been performed with conventional partially N-acetylated chitosans. Herein, the purpose was to identify fundamental chitosan molecular properties favoring siRNA delivery and efficient gene silencing in mammalian cells. Nanoparticles were prepared from well-defined chitosans of various chemical compositions, degrees of polymerization (DPn) and chain architectures. Structure-activity relationships were determined by the cellular uptake of siRNA and the knockdown efficiency at mRNA and protein levels. Additionally, the nanoparticle cytotoxicity was evaluated on the basis of cellular metabolic activity and membrane integrity. Our results show that the most efficient gene silencing was achieved using fully de-N-acetylated chitosans with intermediate chain lengths (DPn 100–300). These chitosans mediated efficient siRNA delivery at low siRNA concentrations and, in several cell lines, potent long-term silencing of both exogenous and endogenous target genes, with minimal cytotoxicity.

Hypoxia-Induced Epigenetic Regulation and Silencing of the BRCA1 Promoter

Disruption of the BRCA1 tumor suppressor can be caused not only by inherited mutations in familial cancers but also by BRCA1 gene silencing in sporadic cancers. Hypoxia, a key feature of the tumor microenvironment, has been shown to downregulate BRCA1 at the transcriptional level via repressive E2F4/p130 complexes. Here we showed that hypoxia also drives epigenetic modification of the BRCA1 promoter, with decreased H3K4 methylation as a key repressive modification produced by the lysine-specific histone demethylase LSD1. We also observed increased H3K9 methylation coupled with decreased H3K9 acetylation. Similar modifications were seen in the RAD51 promoter, which is also downregulated by hypoxia, whereas exactly opposite changes were seen in the promoter of the hypoxia-inducible gene VEGF. In cells containing the BRCA1 promoter driving a selectable HPRT gene, long-term silencing of the promoter was observed following exposure to hypoxic stress. Clones with silenced BRCA1 promoters were detected at frequencies of 2% or more following hypoxia, but at less than 6 × 10(-5) without hypoxia. The silenced clones showed decreased H3K4 methylation and decreased H3K9 acetylation in the BRCA1 promoters, consistent with the acute effects of hypoxic stress. Hypoxia-induced BRCA1 promoter silencing persisted in subsequent normoxic conditions but could be reversed by treatment with a histone deacetylase (HDAC) inhibitor but not with a DNA methylation inhibitor. Interestingly, treatment of cells with inhibitors of poly(ADP-ribose) polymerase (PARP) can cause short-term repression of BRCA1 expression, but such treatment does not produce H3K4 or H3K9 histone modification or BRCA1 promoter silencing. These results suggest that hypoxia is a driving force for long-term silencing of BRCA1, thereby promoting genome instability and tumor progression.

Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications

Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (ε-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter ∼ 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was ∼ 89.3–97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (∼40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications.

Abstract 4076: Biology of altered choline metabolism in ovarian cancer: Prognostic and therapeutic opportunities

Abstract Epithelial Ovarian Cancer (EOC) remains the leading cause of death in women with gynecologic malignancies, due to late diagnosis and early relapse associated with development of chemoresistance. We recently reported alterations of the choline-metabolite magnetic resonance spectral (MRS) profile in EOC cells, characterized by an increase content of phosphocholine (PCho). A multidisciplinary approach revealed that one of the more relevant changes was a significant increase of Choline Kinase (ChoK) activity (consistent with an increased protein and alpha-isoform mRNA expression) in cancer cells as compared to the normal counterpart. ChoK catalyzes the phosphorylation of choline to yield PCho as the first step in the biosynthesis of phosphatidylcholine, required for mammalian cell structural stability and proliferation. ChoK has been extensively related to cell proliferation being at the cross-roads of signalling pathways related to mitogenesis and survival and a prognostic role for ChoK in some cancer types has been described. Aim of the study is the evaluation of the biological relevance of ChoK expression and activity to define its possible role as a non-invasively detectable EOC biomarker and druggable target. We specifically silenced ChoK-alpha expression by transient RNA interference in two different EOC cell lines and evaluated the biological effects. Following ChoK-alpha silencing we observed a 70% reduction of mRNA and protein expression with a similar reduction in PCho accumulation as assessed by HMRS analysis. The ChoK-alpha silencing was accompanied by 20% inhibition of cell growth and similar percentage of cells blocked in the G1-phase of cell cycle. By transcriptome analysis we found CyclinA, related to regulation of cell cycle progression, and cytokines genes (IL-6 and IL-8) related to inflammation and EOC aggressiveness among the most relevant co-repressed genes, whereas among the most up-regulated genes we found Acyl-CoA synthetase and Osteoprotegerin. These observations suggest ChoK-alpha involvement in cell proliferation and survival. Evaluation of the biological effects of long-term Chok-alpha silencing by lentiviral transduction, together with concurrent inhibition of relevant survival signaling pathway (PI3K/AKT) or other enzymes involved in PCho metabolism (PC-PLC) is currently ongoing. To evaluate its possible role as prognostic biomarker, ChoK-alpha mRNA and protein expression is currently being analyzed on well characterized retrospective EOC case materials. The assessment of biological and prognostic relevance of choline metabolites might significantly contribute in the identification of new non-invasive diagnostic modalities and in the development of new targeted therapeutic approaches. (Partially supported by AIRC) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2011-4076