Non-targeting siRNA Research Articles

Abstract 2671: BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation

Abstract The combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) has been FDA approved to treat melanomas harboring (V600E) mutation. Although most BRAF mutant melanomas are highly responsive to these treatments, they all relapse ultimately. In this study, we found a unique mechanism which can be used to treat BRAFi resistant patients. Five BRAFi resistant (BR) cells were established from known BRAF mutant cell lines A375, MEL-1220, A2058, UACC-62, and SK-MEL28 by long-term exposure to BRAFi. These BR cells are hypersensitive to ADI-PEG20, an enzyme which degrades arginine to citrulline. ADI-PEG20 treatment resulted in a 2-3-fold decrease in BR cell viability (MTT assay) and 10-30% increase in apoptosis (Annexin V/PI) in BR cells compared to their paternal cells. The mechanisms involved are as follows: All BR cells express very low levels or do not express argininosuccinate synthetase (ASS, a key enzyme to generate arginine from citrulline). Thus, these BR cells rely primarily on exogenous arginine. Furthermore, these BR cells also have attenuation of glucose uptake which makes them rely mainly on amino acids. Importantly, their ability to undergo autophagy upon nutritional stress is also impaired. The underlying mechanism for low levels of ASS is due to diminished c-Myc expression which is a positive regulator for ASS. Additionally, AMPK-α1, which possesses phosphorylation site at Thr172 governing autophagy and glucose uptake, was attenuated in BR cells. Overexpression of AMPK-α1 using plasmid transfection in A2058BR and MEL-1220BR cells rescued 8-28% ADI-PEG20-induced apoptosis and enhanced autophagosome formation (detected by LysoTacker). Conversely, knockdown of AMPK-α1 using siRNA significantly enhanced ADI-PEG20-reduced cell viability (compared to non-targeting siRNA) in A2058 and MEL-1220 cells through attenuation of autophagy and glucose uptake. This is evidenced by decreased GLUT1 and LC3-II expression, and low activity of 2-NBDG uptake, which also can be seen in most BR cells. The xenograft model also showed that ADI-PEG20 aborted tumor growth of A2058BR and A375BR cells but retarded growth of A2058 and A375 cells. Immunohistochemical staining further confirmed lower levels of ASS and AMPK-α1 in A2058BR xenograft and in tumor tissues from 10 BR patients relative to their parental counterparts (average H-scores of ASS and AMPK in parental tissues vs. BR tissues are 58.2 vs. 7.8, and 146 vs. 78.3, respectively, p <0.03). Importantly, these findings also apply to cell lines and tumor samples from patients who failed both BRAF and MEK inhibitor. In summary, our data suggest that attenuated AMPK-α1 mediated autophagy and glucose uptake and decreased c-Myc mediated ASS re-expression sensitize BR cells to arginine deprivation. Thus, ADI-PEG20 is a potential salvage therapy for BR patients (Supported by R01CA152197). Citation Format: Ying-Ying Li, Chunjing Wu, Sumedh Shah, Shu-Mei Chen, Medhi Wangpaichitr, Lynn Feun, Macus Kuo, Miguel Suarez, Niramol Savaraj. BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2671.

52 Altered ACVR2A expression modifies the response of vascular endothelial cells to normotensive and preeclamptic activin a concentrations

Introduction Circulating activin A concentrations are significantly higher in pre-eclampsia (PE) compared with those in a normotensive pregnancy. We and others have previously identified ACVR2A, which codes for the activin A binding receptor, as a maternal PE susceptibility gene with reduced expression in PE. The PE-associated rs1424954 genetic polymorphism also results in decreased receptor expression. Objective Given that PE is characterised by systemic vascular endothelial dysfunction, we used an in vitro cell-line model to investigate how reduced ACVR2A expression modifies the response of maternal vascular endothelial cells to normotensive and PE activin A concentrations. Methods The SGHEC-7 cell line was used as a model of vascular endothelial cells. ACVR2A expression was silenced using siRNA transfection and confirmed with real-time PCR and immunoblotting. A non-targeting siRNA was used as a negative control (NC). Following a 72 h transfection, SGHEC-7s were treated for 24 h with activin A concentrations representative of normotensive (0, 10 ng/ml) and PE (50 ng/ml) pregnancies. SGHEC-7 proliferation was determined using the electrical impedance-based xCELLigence system, while permeability was measured with a Millipore fluorescence-based assay. Student’s t test and one-way ANOVA with Bonferroni’s post-hoc test where appropriate were used for statistical evaluations. Each experiment was performed at least n = 3 times. A p -value of Results ACVR2A siRNA markedly reduced ACVR2A mRNA and protein levels by 78% ( p p p p p p p Conclusions This study demonstrates that decreased expression of the ACVR2A receptor, in addition to activin A, can also result in dysfunctional vascular endothelial proliferation and permeability. Therefore, the altered expression of the ACVR2A receptor, resulting from a genetic predisposition, may modify the threshold required for activin A to impact vascular endothelial function, thereby exacerbating systemic vascular endothelial dysfunction in PE.

BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

Systemic delivery of siRNA by aminated poly(α)glutamate for the treatment of solid tumors

Small interfering RNA (siRNA) can silence the expression of a targeted gene in a process known as RNA interference (RNAi). As a consequence, RNAi has immense potential as a novel therapeutic approach in cancer targeted therapy. However, successful application of siRNA for therapeutic purposes is challenging due to its rapid renal clearance, degradation by RNases in the bloodstream, poor cellular penetration, immunogenicity and aggregation in the blood. In addition, the few oligonucleotide-based nanomedicines that reached clinical trials either go to the liver following systemic administration or are applied topically. Treatment of solid tumors requires selective distribution of siRNA to the target tissue, hence there is an unmet medical need for an efficacious and safe nano-sized delivery system for their clinical use. To overcome these hurdles, we have designed, synthesized and physico-chemically characterized a novel nanocarrier based on aminated poly(α)glutamate (PGAamine). This cathepsin B-biodegradable polymer interacts electrostatically with the siRNA to form a nano-sized polyplex stable in plasma. Treatment with PGAamine-Rac1 siRNA polyplex (siRac1-polyplex) caused specific gene silencing by 80% in HeLa and SKOV-3 human ovarian adenocarcinoma cells as opposed to PGAamine-control non-targeting siRNA polyplex (siCtrl-polyplex) leading to inhibition of cell migration and wound healing abilities. A stepwise dose escalation was performed in order to determine the in vivo maximum tolerated dose (MTD). This was followed by intraperitoneal administration of siRac1-polyplex to mCherry-labeled ovarian adenocarcinoma-bearing mice leading to preferred tumor accumulation of siRac1 (8-fold) which resulted in 38% Rac1 knockdown. Furthermore, the polyplex was administered intravenously to lung carcinoma-bearing mice in which it caused 33% Rac1 knockdown. These promising results led to efficacy studies administering systemic treatment with an anticancer siRNA, siPlk1-polyplex, which inhibited tumor growth by 73% and 87% compared with siCtrl-polyplex or saline-treated mice, respectively, leading to prolonged overall survival. These findings represent the first time that a polyaminated poly(α)glutamate polymer is used for an efficacious and safe tumor delivery of RNAi following systemic administration.

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice.

Despite their antiviral effect, the invivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. Inexplants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3-4weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

Inhibition of suppressor of cytokine signaling 1 on lipopolysaccharide-induced mucin5AC hypersecretion and the mechanism in human bronchial epithelial cells

To explore the inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in lipopolysaccharide (LPS)-induced mucin5AC (MUC5AC) hypersecretion and the potential mechanism involved in this process. The human bronchial epithelial cells 16HBE were divided into 0, 0.5, 1, 6, 12 and 24 h groups according to the time of LPS challenge. In gain- and loss- of functions experiments, wild-type SOCS1 and SOCS1-targeted siRNA (SOCS1-siRNA) were synthesized to identify the function of SOCS1 in LPS-mediated MUC5AC hypersecretion, and named wild-type SOCS1 group and SOCS1-siRNA group, respectively, and the non-transfected group and non-targeted siRNA group were used as controls. In Filgotinib group, the specific inhibitor of Janus kinase 1 (JAK1), Filgotinib, was used to detect the role of JAK1/signal transducer and activator of transcription 1 (STAT1) signaling pathway in LPS challenge, and the aqueous physiological buffer group was used as the control. The production of MUC5AC protein was measured by enzyme linked immunosorbent assay (ELISA), and the amount of MUC5AC protein was normalized to the total protein in cell lysates and was expressed as μg/mg cell lysates. The proteins expressions of SOCS1, phosphorylation of JAK1 and STAT1 were measured by Western blot, and the total expression of its protein (for JAK1 and STAT1) or β-actin (for SOCS1) was used as the loading control. Compared to 0 h group, LPS induced a robust induction in MUC5AC expression, the expression levels of MUC5AC in 0, 0.5, 1, 6, 12 and 24 h groups were (2.86±0.20), (3.42±0.29), (3.43±0.12), (10.22±0.96), (14.56±1.12), (14.15±1.34) μg/mg, in association with a decrease of SOCS1 expression. And in 6 h group, the expressions of MUC5AC and SOCS1 were both medium up-regulated (all P<0.05). Consequently, the application of LPS for 6 h was selected as the optimal responses period in the ensuing experiments. Compared to the expression of MUC5AC protein in non-transfected group, high level of SOCS1 in wild-type SOCS1 group led to a reduced phosphorylation of JAK1/STAT1, as well as Filgotinib did, thereby suppressing excessive MUC5AC production in wild-type SOCS1 group and Filgotinib group [(4.04±0.65), (7.02±0.83) vs (10.37±1.00) μg/mg] (all P<0.05). Conversely, compared to the expression of MUC5AC in non-targeted siRNA group, down-regulation of SOCS1 in SOCS1-siRNA group promoted the phosphorylation of JAK1/STAT1, and then further increased MUC5AC production [(13.69±1.32) vs (11.01±1.41) μg/mg] (all P<0.05). These results show that SOCS1 suppresses LPS-promoted MUC5AC hypersecretion through the inhibition of JAK1/STAT1 signaling pathway.

MP84-09 INTERFERENCE WITH GLI BINDING TO ANDROGEN RECEPTORS ALTERS THE AR TRANSCRIPTOME AND EFFECTIVELY INHIBITS ANDROGEN GROWTH-INDEPENDENCE OF PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-09 INTERFERENCE WITH GLI BINDING TO ANDROGEN RECEPTORS ALTERS THE AR TRANSCRIPTOME AND EFFECTIVELY INHIBITS ANDROGEN GROWTH-INDEPENDENCE OF PROSTATE CANCER CELLS Na Li, Sarah Truong, Mannan Nouri, Amy Lubik, Josselin Caradec, and Ralph Buttyan Na LiNa Li More articles by this author , Sarah TruongSarah Truong More articles by this author , Mannan NouriMannan Nouri More articles by this author , Amy LubikAmy Lubik More articles by this author , Josselin CaradecJosselin Caradec More articles by this author , and Ralph ButtyanRalph Buttyan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2233AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Progression to castration resistant prostate cancer (CRPC) is associated with a change in the AR-driven transcriptome away from genes associated with prostate epithelial differentiation towards genes associated with cell survival and mitosis. Previously we showed that Gli proteins bind to the enigmatic N-terminal tau5 transactivation domain of AR and co-activate full-length and truncated ARs. When exogenously overexpressed in androgen growth-dependent LNCaP cells, Gli proteins can elicit androgen growth-independence (AI). Here we show that interference with Gli-AR binding in AI LNCaP variants suppresses the alternate AR transcriptome, restores the AR differentiation transcriptome and suppresses AI growth. METHODS Androgen growth-independent, enzalutamide- (ENZ-) resistant LNCaP cell variants, LNCaP-AI and LN95, were treated with Gli inhibitors (GANT61 and HPI-1) or transfected with Gli3 siRNA or transfected with a small decoy peptide from the Gli2 AR-binding domain (Gli-DP) to disrupt intracellular Gli-AR interactions. RNAs were extracted and expressions of differentiation genes (DGs), PSA and KLK2 or mitotic-regulatory genes (MRGs), UBE2C, CDK1 and CDC20 were measured by real-time qRT-PCR and compared to control-treated (vehicle, non-targeting siRNA or empty vector transfected) cells. Cell growth after Gli manipulation(s) was measured on the Incuyte-Zoom instrument that quantifies real-time cell growth and was compared to control cells. Increasing doses of Enzalutamide (ENZ) were titrated into Gli-DP transfected cells to test whether it cooperates with Gli-AR binding disruption in suppressing AI growth. RESULTS Suppression of global Gli activity, Gli3-specific knockdown and exogenous Gli-DP expression significantly suppressed expressions of MRGs but increased expressions of DGs in both AI cell types. These actions also significantly slowed growth of the cells, with Gli inhibitors and Gli3 knockdown showing complete growth suppression whereas the Gli-DP achieved half-suppression. ENZ treatments further increased growth suppression of the Gli-DP expressing cells. CONCLUSIONS Our results identify the potential for Gli-AR binding interference as a means to control AI/CRPC growth. This interference further cooperates with ENZ to provide very robust growth suppression showing that combined targeting of AR N-terminal and C-terminal domains holds the key for more effective treatments for CRPC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1092-e1093 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Na Li More articles by this author Sarah Truong More articles by this author Mannan Nouri More articles by this author Amy Lubik More articles by this author Josselin Caradec More articles by this author Ralph Buttyan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.

Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis

There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye.

Downregulation of LSD1 suppresses the proliferation, tumorigenicity and invasion of papillary thyroid carcinoma K1 cells.

The present study aimed to evaluate the effects of lysine-specific demethylase 1 (LSD1) downregulation, induced by small interfering RNA (siRNA) transfection, on the proliferation, colony formation, migration and invasion of the papillary thyroid carcinoma K1 cell line. The siRNA targeting LSD1 and scrambled non-targeting siRNA were each transfected into papillary thyroid carcinoma K1 cells. Downregulation of LSD1 mRNA and protein level was evaluated by reverse transcription-quantitative polymerase chain reaction, and immunocytochemical (ICC) analysis and western blotting, respectively. A Cell Counting kit-8 assay was applied to estimate the effect of LSD1-siRNA on cell growth. Migration and invasion abilities were estimated by Transwell chamber assay. A soft agar colony formation assay was performed to estimate the effect of LSD1-siRNA on tumorigenicity in vitro. ICC data showed that LSD1 protein was strongly expressed in the blank and control K1 cells compared with the LSD1-siRNA cells (F=15.192, P<0.01). Compared with the control cells, cells transfected with siRNA targeting LSD1 exhibited significant downregulation of LSD1 mRNA (t=6.845, P<0.01) and protein (F=53.764, P<0.01) levels. siRNA targeting LSD1 also downregulated cell proliferation following transfection for 24, 48 and 72 h (t=4.777, P<0.001; t=3.302, P=0.003; and t=3.017, P=0.006, respectively). Compared with the control group, the amount of cell invasion was gradually reduced in the LSD1-siRNA group (t=12.301, P<0.01). The number of migrating cells was significantly higher in the negative control group compared with the LSD1-siRNA group (t=7.911, P<0.01), and the ability of colony formation in the LSD1-siRNA cells was notably reduced in the soft agar formation assay (t=3.612, P=0.005). siRNA targeting LSD1 efficiently inhibits the proliferation, colony formation, migration and invasion of papillary thyroid carcinoma K1 cells.

ERK1/2 MAPK signaling in hypothalamic paraventricular nucleus contributes to sympathetic excitation in rats with heart failure after myocardial infarction.

Brain MAPK signaling pathways are activated in heart failure (HF) induced by myocardial infarction and contribute to augmented sympathetic nerve activity. We tested whether decreasing ERK1/2 (also known as p44/42 MAPK) signaling in the hypothalamic paraventricular nucleus (PVN), a forebrain source of presympathetic neurons, would reduce the upregulation of sympathoexcitatory mediators in the PVN and augmented sympathetic nerve activity in rats with HF. Sprague-Dawley rats underwent left anterior descending coronary artery ligation to induce HF, with left ventricular dysfunction confirmed by echocardiography. One week after coronary artery ligation or sham operation, small interfering (si)RNAs targeting ERK1/2 or a nontargeting control siRNA was microinjected bilaterally into the PVN. Experiments were conducted 5-7 days later. Confocal images revealed reduced phosphorylated ERK1/2 immunofluorescence in the PVN of HF rats treated with ERK1/2 siRNAs compared with HF rats treated with control siRNA. Western blot analysis confirmed significant reductions in both total and phosphorylated ERK1/2 in the PVN of HF rats treated with ERK1/2 siRNAs along with reduced expression of renin-angiotensin system components and inflammatory mediators. HF rats treated with ERK1/2 siRNAs also had reduced PVN neuronal excitation (fewer Fos-related antigen-like-immunoreactive neurons), lower plasma norepinephrine levels, and improved peripheral manifestations of HF compared with HF rats treated with control siRNAs. These results demonstrate that ERK1/2 signaling in the PVN plays a pivotal role in mediating sympathetic drive in HF induced by myocardial infarction and may be a novel target for therapeutic intervention.

HNF1 regulates critical processes in the human epididymis epithelium

The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1α and HNF1β transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment.

The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells.

BackgroundRNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a “noncoding RNAi”. However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen).ResultsCompared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response.ConclusionsSignificant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and “non coding” siRNA is a hindrance in the development of siRNA based therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2267-9) contains supplementary material, which is available to authorized users.

Targeting the X-Linked Inhibitor of Apoptosis Protein (XIAP) Can Promote Tumor Cell Death, and Increase the Cytotoxic Effects of Chemotherapy Agents in in Vitro and In Vivo Models of Rituximab-Resistant Lymphoma

In diffuse large B-cell lymphoma (DLBCL) the addition of rituximab to front line multi-agent therapy has changed the biology and clinical outcome of patients with relapsed/refractory disease. To better understand the mechanisms responsible for rituximab/chemotherapy cross-resistance we developed several rituximab resistance cell lines, which also display significant resistance to a wide range of chemotherapy agents. These therapy resistant cell lines (TRCL)s have multiple deregulations the apoptotic response pathway, including a loss of Bak and Bax. We previously demonstrated that small molecule mimetics of the second mitochondrial activator of caspases (SMAC), which antagonizes the inhibitor of apoptosis (IAP) family of proteins, could boost response to chemotherapy agents and extend survival in TRCL xenograft animal models. To investigate how SMAC mimetics overcome chemotherapy resistance in TRCL (RL-4RH and Raji-4RH) models we used RNA interference to transiently knockdown expression of individual IAPs (cIAP1, cIAP2, livin, survivin, and XIAP), and then exposed those cells to a panel of chemotherapy agents. Apoptosis induction was analyzed by flow cytometry staining for annexin V and Sytox blue (a DNA stain). While SMAC mimetics can decrease levels of the cellular inhibitor of apoptosis proteins (cIAP1/cIAP2), siRNA knockdown of cIAP1/cIAP2 did not increase chemotherapy-induced apoptosis. On the other hand, siRNA knockdown of XIAP in TRCLs substantially increased rates of apoptosis compared to cells transfected with non-targeting siRNA (5% in controls vs. 22% in XIAP knockdown RL-4RH cells). An additional 24% of XIAP knockdown RL-4RH cells showed signs of necrosis compared to 0.5% in control RL-4RH. Results were confirmed by western blot (PARP cleavage). In addition, siRNA XIAP knockdown re-sensitized TRCLs (Raji-4RH and RL-4H) to the cytotoxic effects of multiple chemotherapy agents (gemcitabine, etoposide, and vincristine). To investigate if XIAP knockdown could improve chemotherapy response in vivo we created a TRCL (Raji-4RH) that stably expresses XIAP targeting shRNA along with luciferase to enable in vivo imaging. SCID mice were inoculated with either the Raji-4RH XIAP knockdown or Raji-4RH non-targeting shRNA control cell line (10x106 cells per animal given i.v.). Tumors were allowed to engraft for 7 days prior to treatment administration. Animal were either given no treatment, or rituximab in combination with ifosfamide, carboplatin, etoposide (RICE) and mesna. Following treatment animals were imaged on a weekly basis to track tumor progression. At the time of the submission disease progression was present in animals in the observation groups, and in animals inoculated with non-targeting control shRNA Raji-4RH treated with RICE. InterestingHowever, animals inoculated with XIAP knockdown Raji-4RH and treated with RICE therapy remain tumor free with no detectable disease. In summary, XIAP acts as a key cell survival regulator in rituximab-chemotherapy resistant lymphoma models. Downregulation of XIAP can enhance chemotherapy activity and promote tumor cell death. Additionally, XIAP knockdown appears to enhance the activity of RICE, a commonly used regiment in the relapsed/refractory setting for DLBCL patients. Our results support the hypothesis that selective XIAP inhibitors, currently in development, may be highly active against relapsed/refractory B-cell lymphoma. DisclosuresCzuczman:Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board; MorphoSys: Consultancy; Celgene: Employment.

4 SUPPRESSION OF ASH2L ALTERS DNA METHYLATION AND HISTONE PATTERNS DURING BOVINE EMBRYONIC DEVELOPMENT

Epigenetic patterns established during early bovine embryogenesis via DNA methylation and histone modification patterns are essential for proper gene expression and embryonic development. We have previously discovered that suppression of absent, small, or homeotic-like (ASH2L) with small interfering RNA (siRNA) had no significant effect during in vitro embryo development when compared with its respective control (31.3 ± 2.0% standard error of the mean, n = 466 v. 34.8 ± 1.9%, n = 418). Analysing DNA methylation and histone modifications via immunocytochemistry will further explain the role of ASH2L during embryonic development, specifically at the blastocyst stage. In this experiment, we obtained mature bovine oocytes from a commercial supplier (De Soto Biosciences, Seymour, TN) and preformed IVF following standard laboratory protocol. Eighteen hours after IVF, presumptive zygotes were divided into 3 treatments: noninjected controls, nontargeting siRNA injected controls (siNULL), and injection with siRNA targeting ASH2L (siASH2L). Each embryo was injected with ~100 pL of 20 nM siRNA previously verified to suppress expression of ASH2L by ~79%. Embryos were cultured in Bovine Evolve (Zenith Biotech, Guilford, CT) supplemented with 4 mg mL–1 of BSA (Probumin, Millipore) for 7 days. Blastocysts from each treatment (N = 601) were fixed and prepared for immunocytochemistry following standard laboratory protocol. The following primary antibodies were used to target specific DNA and histone methylation marks: 5mc mAb (Epigentek, Farmingdale, NY), 5hmc pAb, H3K4me3 pAb (Active Motif, Carlsbad, CA), H3K4me2 pAb, H3K9me2–3 mAb, and H3K27me3 mAb (Abcam, Cambridge, MA). Embryos were fluorescently labelled with the following secondary antibodies: Alexa Flour 488 Goat Anti-Rabbit, Alexa 488 Donkey Anti-Goat, and Alexa Flour 594 Goat Anti-Mouse (Invitrogen, Carlsbad, CA). The DNA was stained with Hoechst 33342 (Invitrogen). Fluorescent images were captured using the Zeiss Stallion digital imaging work station. Ratio averages (targeting mark/DNA) were calculated and statistical analysis performed using one-way ANOVA and Tukey’s honestly significant difference to assess treatment effects. The ratio of DNA methylation to total DNA increased in siASH2L as compared with control and siNULL embryos (0.35 ± 0.01, 0.26 ± 0.02, and 0.30 ± 0.01, respectively; P &lt; 0.01). The 5hmC was inversely related to 5mC levels and decreased in siASH2L embryos (0.75 ± 0.01, 0.93 ± 0.02, 0.87 ± 0.02, respectively; P &lt; 0.0001). The H3K4me3 and H3K27me3 are also inversely related with decreased H3K4me3 in siASH2L versus control and siNULL embryos (0.48 ± 0.02, 0.57 ± 0.02, 0.58 ± 0.02, respectively; P &lt; 0.001) and increased H3K27me3 (0.62 ± 0.02, 0.053 ± 0.01, 0.54 ± 0.02, respectively; P &lt; 0.001). No differences were observed in H3K9me2–3 or H3K4me2 labelling across treatments. These results indicate that ASH2L may play a role in DNA methylation by decreasing 5mc and 5hmc conversion, which is a key event during early embryonic development. Suppression of ASH2L also alters global levels of H3H4me3 and H3K27me3, which may lead to transcription aberrations. Further analysis of siASH2L embryos via RNA-seq will help define its role during early embryonic development.

Abstract C138: Targeting macropinocytosis in pancreatic cancer

Abstract Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States with a 5-year survival rate of &amp;lt;5%. The deadly nature of this disease compounded by a lack of effective treatment options highlights a need for new therapeutics. Past studies have suggested that pancreatic cancer cells acquire the nutrients needed for cell growth through macropinocytosis, an endocytic process by which protrusions from the cell membrane create large vesicles that transport extracellular fluid along with the constituent proteins and lipids into the cell. Additionally, several studies have suggested that macropinocytosis is regulated in part by various genes including p21 protein (Cdc42/Rac)-activated kinase1 (PAK1), ADP-ribosylation factor 6 (ARF6), and sorting nexin 5 (SNX5). In this study, we investigated the effects of knockdown of these three genes on the macropinocytic activity of pancreatic cancer cells. The goal of this study is to assess whether PAK1, ARF6, or SNX5 may serve as potential targets for the suppression of macropinocytosis in pancreatic cancer, which would hamper the cells' uptake of nutrients thus starving the cancer cells and hindering tumor growth. We carried out this study using MIA PaCa-2 pancreatic cancer cells, which are known to exhibit relatively high levels of macropinocytosis. Treatment of cells with siRNA sequences specific to the three genes resulted in a significantly decreased uptake of FITC-labeled dextran as compared to cells treated with non-targeting siRNA and cells that received no treatment, indicating that inhibiting the expression of each of the genes correlates to a reduction in macropinocytic activity. Among the three genes, PAK1 siRNAs exhibited the most significant and consistent inhibitory effect on macropinocytic activity of MIA PaCa-2 cells. We, therefore, further tested the effect of two small-molecule PAK1 inhibitors, namely IPA-3 and PF-03758309, on macropinocytosis. Treatment of cells with the inhibitors resulted in a dose-dependent decrease in the uptake of FITC-labeled dextran and FITC-labeled albumin. The inhibitors also showed potent anti-proliferative activity in pancreatic cancer cell lines. The results of this study indicate that inhibition of PAK1, ARF6, or SNX5 all resulted in reduced macropinocytic activity of pancreatic cancer cells, with PAK1 showing the most promise as a potential therapeutic target for pancreatic cancer. Citation Format: James Hunt, Serina Ng, Clifford Whatcott, Daniel D. Von Hoff, Haiyong Han. Targeting macropinocytosis in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C138.

Down-regulation of astroglial glutamate transporter-1 in the locus coeruleus impairs pain-evoked endogenous analgesia in rats

Descending noradrenergic inhibition to the spinal cord from the locus coeruleus (LC) is an important endogenous pain-relief mechanism which can be activated by local glutamate signaling. Here we tested whether dysregulation of extracellular glutamate level in the LC induced by down-regulating astroglial glutamate transporter-1(GLT-1) impairs endogenous analgesia. In rats treated with repeated LC injections of GLT-1 selective or non-targeting small interfering RNA (siRNA), a subdermal injection of capsaicin was used to examine noxious stimulation-induced analgesia (NSIA), evoked LC glutamate and spinal noradrenaline release, and evoked LC neuronal activity. LC-injected GLT-1 siRNA reduced expression of GLT-1 in the LC (P=0.02), increased basal activity of LC neurons (P<0.01), and increased basal extracellular concentrations of LC glutamate (P<0.01) and spinal noradrenaline (P<0.01), but did not affect mechanical withdrawal thresholds in the hindpaw (P=0.83), compared to non-targeting siRNA. LC-injected GLT-1 siRNA impaired capsaicin-evoked release of LC glutamate and spinal noradrenaline, capsaicin-evoked LC neuronal activation, and NSIA. These results suggest that astroglial GLT-1 is essential to normal LC function and that increased extracellular glutamate by down-regulating GLT-1 impairs evoked LC activity and NSIA, essentially taking the LC “off-line”.

Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway.

The bioactive steroid, marinobufagenin, is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthesized by adrenocortical and placental cells. Marinobufagenin binding to Na/K-ATPase initiates profibrotic cell signaling, and heightened marinobufagenin levels are implicated in the pathogenesis of hypertension, preeclampsia, and chronic kidney disease. Steroids are derived from cholesterol through the traditional steroidogenesis pathway initiated by enzyme CYP11A1, and via the acidic bile acid pathway, which is controlled by enzyme CYP27A1. The mechanism of marinobufagenin biosynthesis in mammals, however, remains unknown. Here, we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and marinobufagenin levels by 67% when compared with nontreated cells or cells transfected with nontargeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect marinobufagenin production in either cell culture, but suppressed production of progesterone 2-fold in human trophoblast cells and of corticosterone by 90% in rat adrenocortical cells when compared with cells transfected with nontargeting siRNA. In vivo, in a high-salt administration experiment, male and female Dahl salt-sensitive rats became hypertensive after 4 weeks on a high-NaCl diet, their plasma marinobufagenin levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold and 2.0-fold. Therefore, the endogenous steroidal Na/K-ATPase inhibitor, marinobufagenin, is synthesized in mammalian placenta and adrenal cortex from cholesterol through the novel acidic bile acid pathway. These findings will help to understand the role of marinobufagenin in highly prevalent human cardiovascular diseases.

Abstract POSTER-THER-1426: Characterization and targeting of TWIST family proteins to prevent chemoresistance and metastasis in ovarian cancer

Abstract INTRODUCTION/PURPOSE: The vast majority of epithelial ovarian cancer (EOC) patients present with advanced metastatic disease, but will respond well to first line chemotherapy consisting of a platinum drug and/or paclitaxel. Unfortunately, most of these patients will relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. Therefore, novel therapies that can eliminate disseminated and chemoresistant cells are urgently needed. Two potential targets to supplement current treatment are TWIST1 and TWIST2, related basic helix-loop-helix transcription factors vital in development and reactivated in many cancers. TWIST proteins regulate epithelial to mesenchymal transition (EMT), the process underlying metastatic spread. Furthermore, recent studies from multiple groups have correlated mesenchymal characteristics with a cancer stem cell phenotype, and with chemoresistance to a variety of agents. However, transcription factors are difficult to target with small molecule drugs due to their nuclear localization. Therefore, we employ siRNA to target TWIST mRNA, in order to reduce metastasis and re-sensitize cancer cells to conventional chemotherapeutic agents. METHODS: We have designed and validated two siRNAs against TWIST. We have also developed two nanoparticle-based delivery systems for siRNA which would be translatable from the bench to the clinic. The first is YTZ3-15, an amphiphilic generation three poly(amidoamine) (PAMAM) dendrimer, the second, a polyethylenimine (PEI) coated mesoporous silica nanoparticle (MSNP). Several EOC cell lines varying in their TWIST1 and TWIST2 expression were treated siRNA delivered via YTZ3-15 or MSNPs. Fluorescent microscopy was used to evaluate siRNA uptake. Western blotting and qRT-PCR were used to assay TWIST knockdown. Sulphorhodamine B and MTT cytotoxicity assays were used to test chemosensitivity before and after TWIST knockdown. TWIST1 levels in clinical samples were evaluated using IHC. RESULTS: Fluorescent microscopy demonstrated that all tested cell lines efficiently take up PAMAM dendrimers and MSNPs, delivering their siRNA cargo to the cell interior. Tracking of punctate signals over time showed slow release from endosomes into the cytosol. Both YTZ3-15 and MSNP delivery of siRNA resulted in robust TWIST knockdown lasting at least one week post-transfection. Furthermore, TWIST knockdown sensitized cells to chemotherapy compared to cells treated with non-targeting control siRNA. In clinical samples, nuclear TWIST1 staining correlated with tumor stage, with early stage tumors showing cytoplasmic staining, and staining in surrounding cells rather than tumor cells themselves. CONCLUSIONS: TWIST family proteins are promising targets to address the compound problems of metastasis and acquired drug resistance in EOC. Addition of TWIST inhibitors, such as the siRNA-based technologies we have developed, to a traditional treatment regimen has the potential to cure currently intractable disease. These combined therapies will drastically improve survival and provide new hope for patients with recurrent ovarian cancer. Citation Format: Cai Roberts, Gina Lowe, James Finlay, Joana Loeza, Thanh Dellinger, Ernest Han, Jeff Zink, Fuyu Tamanoi, Carlotta Glackin. Characterization and targeting of TWIST family proteins to prevent chemoresistance and metastasis in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1426.

Abstract 3789: BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule (MT)-depolymerizing agent with a broad in vitro anti-proliferative activity against human tumor lines refractory to standard MT-targeting agents. The prodrug has anti-cancer activity in diverse tumor models when given orally or iv; alone or in combination therapy. In this study, the role of the ‘Spindle Assembly Checkpoint’ (SAC) in the mechanism of action of BAL27862 was evaluated. Methods: To evaluate BAL27862-resistance mechanisms, tumor lines were selected in vitro by increasing drug levels. Anti-proliferative activity was analyzed using Crystal Violet assay. siRNA transfection for 24 h was followed by drug treatment (50 nM) for 48 h. Mitotic index was evaluated after 18 h treatment by p-Histone H3 staining (pHH) or flow cytometry (FC). SAC complex formation was assessed by BubR1 immunoprecipitation (IP) and immunoblotting for SAC components. Results: The SAC ensures cell cycle arrest upon spindle disruption, promoting tumor cell death after prolonged mitotic arrest. SAC formation involves phosphorylation and assembly of a multiprotein complex, of which BubR1 and Mad2 are essential components. Using siRNA approaches, BubR1 was shown to be required for BAL27862 anti-proliferative activity in 5 BAL27862 sensitive tumor lines (IC50s: 8-20 nM). A similar analysis of Mad2 in 3 lines resulted in almost identical attenuation of the effect of BAL27862. For example, non-targeting control siRNA (NTC)-treated H460 lung cancer cells entered a cell death program following BAL27862 treatment (∼45% cell death vs. starting cell number); a response attenuated by BubR1 and Mad2 down regulation (71% and 62% proliferation resp. vs. DMSO controls). The accumulation of mitotic arrested (pHH positive) cells associated with BAL27862 treatment was also absent after BubR1 or Mad2 siRNA transfection. To investigate whether BAL27862 (50 nM, 18 h) induces the formation of the SAC, BubR1 IPs from 3 sensitive tumor lines (IC50: HeLa cervical: 20 nM; A549 lung: 15 nM; SKOV3 ovarian: 12 nM) were analyzed for SAC components. In all cases, complex formation was observed, consistent with a concomitant mitotic accumulation as measured by FC. Strikingly, in variants of the same lines selected for BAL27862 resistance (resistance factors: 3.2-9.4) no SAC formation or mitotic accumulation was observed under the same conditions. Moreover, BAL27862-induced protein mobility shifts, consistent with activating phosphorylation of SAC components, were also absent in the resistant variants. Conclusions: The association of SAC activation with BAL101553sensitivity suggests prediction of cancer response may lie in its tumor checkpoint control function. BAL101553 is currently in Phase 2a clinical evaluation in advanced cancer patients. Exploratory biomarkers are being evaluated to aid selection of patients most likely to respond. Citation Format: Felix Bachmann, Karin Burger, Heidi Lane. BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2015-3789