Fold Induction Research Articles

Stress Mis‐programming of Mesenteric Arterial Smooth Muscle is Suppressed by a‐adrenergic Receptor Blockade

The sympathetic nervous system (SNS) programs rodent resistance artery smooth muscle (RASM) maturation from post‐natal day (PND) 14‐35. We hypothesized that stress hyper‐activation of the SNS would cause developmental mis‐programming of RASM resulting in a hyper‐contractile smooth muscle phenotype and vascular dysfunction. Rats were stressed as neonates by maternal separation (MS) for 3hrs/day (PND2‐14) and as adults by 1hr of restraint (RS). During MS, rat pups were injected IP with a1‐adrenergic blocker Terazosin (0.5mg/kg) or Vehicle. Mesenteric arteries were isolated at PND21, 35 or 3‐24hrs after RS. Smooth muscle contractile genes were measured by qPCR and correlated with functional studies by wire myography. MS accelerated the molecular maturation of RASM measured at PND21 with 2‐4 fold increases in MLCK, myosin phosphatase subunits CPI‐17 (inhibitory subunit) and smMHC as compared to un‐stressed littermates (p<0.05). This correlated with increased force to the a‐agonist phenylephrine and to calcium in permeabilized arteries (p<0.05). These effects were suppressed by treatment with Terazosin (p<0.05). Differences between MS and littermate controls normalized by PND35. RS of adult rats caused a 4‐6 fold induction of MLCK, CPI‐17, and smMHC at 3hrs and this resolved at 24hrs with no interaction between MS and RS. We conclude that stress‐induced SNS hyper‐activation in a critical developmental window or in maturity mis‐programs resistance artery smooth muscle causing vascular dysfunction, but maintained effects during or after maturation likely require additional triggers.

Homocysteine Elicits an Inflammatory Profile in Murine Macrophages Through an EMMPRIN Mediated Pathway

IntroductionMacrophages are known to differentiate between two distinct phenotypic subsets: M1 and M2. The M1 phenotype elicits a pro‐inflammatory profile while M2 macrophages are categorized as being anti‐inflammatory. Hyperhomocysteinemia (HHcy) is associated with many inflammatory diseases such as obesity and atherosclerosis. It is known that HHcy increases specific macrophage secretory factors, such as MCP‐1 and MMP‐9. However, the direct effect of HHcy on macrophage phenotype is largely unexplored. We hypothesize that HHcy causes macrophages to shift into a more inflammatory phenotype or M1.MethodsWe utilized 2 murine macrophage cell lines: Raw 264.7 and J774A.1 cells. These cells were treated with 500 and 100 µM homocysteine, respectively, for 24 hours. Methods employed are Western Blot, ICC, FACS and PCR.ResultsIn Raw 264.7 cells, 24 hour treatment with 500 μM Hcy resulted in a 2.25 fold increase in CD40 protein expression: a cell surface marker on M1 macrophages. ICC confirms this increase in CD40. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) was also increased by 1.62 fold, indicating that MMP‐9 induction with Hcy may be through an EMMPRIN mediated pathway. J774A.1 cells were more responsive to Hcy treatment, demonstrating a 1.4 fold induction of CD40 with 24 hours of 100 μM treatment. ICC demonstrates increased MMP9 production in this cell line. We suggest that Hcy may alter IL‐1β, iNOS, TIMPs 1 and 4, and MMP‐2, as well as EMMPRIN in MMP9 production.ConclusionsOur current data suggests that Hcy treatment shifts macrophage phenotype towards the pro‐inflammatory, M1 subset. We believe that HHcy increases IL‐1 β, iNOS and MMP‐2, while demonstrating decreases in TIMPs 1 and 4.

Different Resistance-Training Regimens Evoked a Similar Increase in Myostatin Inhibitors Expression.

The aim of the present study was to investigate the effect of different resistance-training regimens (S or P) on the expression of genes related to the MSTN signaling pathway in physically-active men. 29 male subjects with at least 2 years of experience in strength training were assigned to either a strength-training group (S; n=11) or a power-training group (P; n=11). The control group (C; n=7) was composed of healthy physically-active males. The S and the P groups performed high- and low-intensity squats, respectively, 3 times per week, for 8 weeks. Muscle biopsies from the vastus lateralis muscle were collected before and after the training period. No change was observed in MSTN, ACTIIB, GASP-1 and FOXO-3 A gene expression after the training period. A similar increase in the gene expression of the inhibitory proteins of the MSTN signaling pathway, FLST (S: 4.2 fold induction and P: 3.7 fold induction, p<0.01) and FL-3 (S: 5.6 fold induction and P: 5.6 fold induction, p<0.01), was detected after the training period. SMAD-7 gene expression was similarly augmented after both training protocols (S: 2.5 fold induction; P: 2.8 fold induction; p<0.05). In conclusion, the resistance-training regimens (S and P) activated the expression of inhibitors of the MSTN signaling pathway in a similar manner.

Caspase-Dependent Anti-Tumor Effects of ONC201/TIC10 on Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM)

PI3K/Akt and Ras/MAPK pathways are attractive therapeutic targets in almost all tumor types, including AML and MM. Apo2L/TRAIL has been deemed a promising therapeutic given its selectivity towards cancer cells although its clinical development has been hampered by various limitations including short half-life and general shortcoming of protein-based therapeutics. ONC201/TIC10 (Oncoceutics, Inc.) is a first-in-class small molecule inducer of TRAIL expression. ONC201/TIC10 has previously been shown to up-regulate TRAIL and its death inducing receptor DR5 in HCT116 colon cancer cells, in part through the inhibition of Foxo3a phosphorylation mediated by dual inhibition of Akt and ERK (Allen JE et al, Sci Transl Med., 2013). Currently, ONC201/TIC10 is set to enter clinical trials for patients with advanced malignancies after the IND was approved by the FDA in March, 2014. We thus investigated the therapeutic potential of ONC201/TIC10 in AML and MM given a major unmet need when conventional therapy fails. We explored the possibility that ONC201/TIC10 induces apoptosis in MM and AML in part through dual inhibition of the PI3K/Akt and Ras/MAPK pathways.We tested a panel of four human MM cell lines (KMS18, MM.1S, MM.1S 33X and RPMI-8226) and three human AML cell lines (Kasumi-1, HL60, HL60/VCR). The Cell-titer Glo assay demonstrated a time and dose-dependent decrease in viability in the entire panel of MM and AML cells. EC50 values ranged from 1-2.5 µM for the MM and 2-5µM for the AML cell lines, respectively. Bortezomib-resistant cells MM.1S 33X and vincristine- resistant cells HL60/VCR were also significantly sensitive to ONC201/ TIC10 as a single agent with EC50s on par with the corresponding parental cell lines. Given the previously reported pro-apoptotic effects of ONC201/TIC10 against solid tumor cells, we assessed apoptosis by performing Sub-G1 analyses and assessing caspase-3 cleavage as two widely used methods to analyze apoptotic cell death. We observed an average of 10-fold induction of ONC201/TIC10–mediated apoptosis in MM cells at 5 mM at 48 hrs post-treatment. Rescue of ONC201/TIC10-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Dose and time dependent induction of apoptosis was noted in western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201/TIC10 for 48 hrs prior to analysis. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously proposed mechanism of ONC201/TIC10 against solid tumor cells. To further investigate the therapeutic potential of ONC201/TIC10 in the context of AML, fresh AML cells were treated with ONC201/TIC10. The primary cells were also found to be sensitive to ONC201/TIC10 (60% decrease in cell viability 72 hrs post 5mM ONC201/TIC10 treatment). Similarly, caspase 3/7 activity was significantly increased as assessed by the Caspase Glo 3/7 assay (~5 fold induction in activity 72 hrs post 5mM ONC201/TIC10 treatment). To explore further the therapeutic potential of ONC201/TIC10, we performed combinatorial experiments with bortezomib and vincristine using the MM.1S 33X MM cells and the HL60/VCR AML cell lines. ONC201/ TIC10 showed an additive effect with both these compounds against the MM and AML lines.Our work demonstrates activity of ONC201/TIC10 against AML and MM cell lines including fresh AML tumor cells. The efficacy data with resistant cells is in par with the applicability of TIC10 in patients with refractory/relapsed hematological malignancies. The long-term goal of this project is to provide a rationale for a phase 1b trial of ONC201/TIC10 for refractory/relapsed MM and AML in combination with existing therapies. [Display omitted] DisclosuresAllen:Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

ATRA-Induced Activation of the Autophagy Regulator Tfeb Regulates Myeloid Differentiation in Acute Promyelocytic Leukemia

IntroductionAcute promyelocytic leukemia (APL) is characterized by gene fusions involving RARα, which disrupt retinoid signaling and lead to the clonal expansion of myeloid precursors halted at the promyelocyte stage of maturation. Treatment with all-trans-retinoic acid (ATRA) can overcome this differentiation block, restore granulocytic differentiation in APL cells and improve APL patient outcome. ATRA treatment of APL cells induces autophagy, a catabolic process whereby redundant proteins and organelles are degraded using lysosomal machinery. ATRA-induced autophagy is important for successful granulocytic differentiation of APL cells through the degradation of fusion oncoproteins and executing the structural protein remodeling necessary for cellular differentiation.The molecular mechanisms linking retinoid signaling and autophagy pathways are poorly understood and are the focus of our study. By integrating available chromatin immunoprecipitation, coupled with next-generation sequencing (ChIPseq) datasets and transcriptomic approaches, we examined the transcriptional effects of ATRA treatment on autophagy-related genes during APL cell differentiation. We identified transcription factor EB (TFEB), a master transcriptional regulator of autophagy, as a retinoid target and have evaluated the consequences of TFEB knockdown on both ATRA-induced autophagy and APL cell differentiation.Methods & ResultsA list of 557 autophagy-related genes was generated from 3 public autophagy databases. Cluster analysis of expression of these 557 genes in the APL TCGA RNAseq dataset, suggest autophagy is associated with patient survival (Cluster 1 = 26.4 months / Cluster 2 = 60 months). We therefore examined whether the 557 autophagy related genes may be direct ATRA targets. Accessing a public ChipSeq database (GSE18886), increased RARα binding was detected within the TFEB gene in NB4 cells (APL cell line, t15:17) treated with ATRA for 24 hours. Increased RNAPolII binding and transcriptionally active histone markers were also detected, consistent with a direct transcriptionally activating retinoid effect.To test the effects of retinoid therapy on APL gene expression, we treated NB4 cells with ATRA or ethanol control for 72 hours. Isolated RNAs were sequenced using the Illumina HiSeq 2000 platform. Reads were aligned and annotated to the hg19 genome using Tophat and subsequent differential expression analysis performed using Cufflinks/Cuffdiff and Cluster/Treeview. We observed changes in autophagy gene expression upon ATRA treatment and recorded a significant >2 fold increase in expression in >50 autophagy-related genes including TFEB, p62 and DRAM1. We then carried out lentivirally-mediated shRNA knockdown of TFEB in NB4 cells and treated both knockdown and scrambled control cells with ATRA for 72 hours. Transcriptomic analysis demonstrated that TFEB knockdown blocked ATRA-induced TFEB induction, affected the regulation of key autophagy-related genes and impaired CD11b expression. These results were validated by qRT-PCR (n=6)(P<0.001).We confirmed TFEB upregulation by qRT-PCR in primary human APL samples treated with ATRA ex vivo (n=1) and in primary peripheral blood mononuclear cells isolated from tumour patients treated with ATRA as part of clinical trial NCT00195156 (n=3). We also found that TFEB was only minimally induced by ATRA in the differentiation-resistant NB4R cell line (qRT-PCR n=6), suggesting an important role for TFEB in ATRA-induced APL differentiation. We evaluated the ability of two clinically used differentiating agents - valproic acid (VPA) and arsenic trioxide (ATO), alone and in combination with ATRA, to induce TFEB expression in NB4 cells. While VPA alone induced TFEB (5 fold), combined therapy with ATRA and VPA resulted in a >20 fold induction of TFEB expression, which correlated with enhanced differentiation (n=6)(P<0.001). ATO alone did not induce TFEB nor did it enhance the induction seen with ATRA. Notably, combined therapy with ATRA and ATO did not significantly enhance differentiation over levels seen with ATRA alone.ConclusionRetinoid signaling promotes APL differentiation in part through transcriptionally activating autophagy. Novel or existing therapies, which enhance autophagy signaling, may promote retinoid-induced differentiation and circumvent resistance to retinoid therapy in acute myeloid leukemia. DisclosuresOrfali:MSD: Unrestricted educational support Other; Amgen: Unrestricted educational support, Unrestricted educational support Other; BMS: Unrestricted educational support, Unrestricted educational support Other; Pfizer: Unrestricted educational support, Unrestricted educational support Other; Novartis: Unrestricted educational support Other.

Microrna Mir-21 Is Upregulated after Different Microenvironmental Stimuli and Controls Proliferation, Chemotaxis and Chemoresistance in Chronic Lymphocytic Leukemia

Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. miR-21 is overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment and metastasis. In CLL, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. In addition, high expression of ZAP-70 protein in CLL is related to shorter overall survival and higher probability of progression. Several biological mechanisms have been described explaining the adverse prognosis associated with high ZAP-70 expression. In this sense, ZAP-70 protein increases the capability of CLL cells to respond to different survival and migration signals provided by the microenvironment. In a previous work, we found that transfected B-cell lines with a ZAP-70 expressing vector (Calpe at al, Blood 2011) had an increased expression of several molecules, including miR-21. Against this background, we studied the relationship between ZAP-70 protein and miR-21. For this, we firstly analyzed the correlation of ZAP-70 with miR-21 in primary CLL cells from 32 patients. In this series we observed that miR-21 expression analyzed by QRT-PCR was significantly higher in patients with high expression of ZAP-70 compared to patients with low expression of ZAP-70 (mean miR-21 in high ZAP-70 (N=17) = 5.781 ± 1.517; mean miR-21 in low ZAP-70 (N=15) = 0.6783 ± 0.2730; p=0.0082). In order to further analyze the molecular mechanisms regulating the induction of miR-21 and the potential role of ZAP-70 protein in this process, we co-cultured primary CLL cells (N=16) in conditions mimicking the microenvironment in the proliferation centers (bone marrow stromal cells with concomitant stimulation of CD40 and TLR9). In these conditions, besides ZAP-70 activation, we observed a 3.6 ± 0.78 mean fold induction in miR-21 expression after 48 hours compared to cells in suspension. Interestingly, this increase was only significant in patients with high expression of ZAP-70 (N=8; p= 0.0379). To define the role of ZAP-70 in miR-21 regulation, we stably transfected Ramos B-cells with ZAP-70 protein and found that both MAPK and STAT3 participate in the induction of miR-21 expression after ZAP-70 activation upon BCR crosslinking. Moreover, using this system we observed downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be targeted by miR-21 in malignant cells. Next, we aimed to study the functional role of miR-21 in primary CLL cells co-cultured in conditions mimicking the microenvironment from the proliferation centers. For this, we analyzed survival, proliferation and response to fludarabine after inhibition of miR-21 expression by transfecting primary CLL cells with antisense miR-21 inhibitor. The co-culture of primary CLL cells significantly increased their survival after 48 hours regardless the inhibition of miR-21. However, the induction of proliferation (measured as percentage of Ki-67 positive cells) was significantly inhibited by the suppression of miR-21 (N=13; p=0.022). Next, the analysis of a small pilot cohort showed a consistent but not yet significant overcoming of the chemoresistance induced by the co-culture after the inhibition of miR-21 Interestingly, the sensitivity to fludarabine of primary CLL cells cultured in suspension was also increased by the inhibition of miR-21. Finally, we examined the ability of primary CLL cells to migrate towards CXCL12 and observed that inhibition of miR-21 resulted in a 2.62-fold reduction of the migration index (ratio of cells migrating towards culture media to cells migrating towards media with 200ng/mL CXCL12). In conclusion, we have showed the correlation and participation of ZAP-70 protein in the regulation of miR-21. We have also observed that miR-21 contributes to CLL proliferation, resistance to fludarabine, and chemotaxis towards CXCL12. Although further experiments are warranted in order to fully elucidate the regulation and functional role of miR-21 in CLL, these results help to enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and could potentially be exploited for the development of new treatments in a near future. DisclosuresBosch:Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Comparative Analysis of Gene Expression Profiling Between Resistant and Susceptible Varieties Infected With Soybean Cyst Nematode Race 4 in Glycine max

Soybean cyst nematode (SCN) is one of the most devastating pathogen for soybean. Therefore, identification of resistant germplasm resources and resistant genes is needed to improve SCN resistance for soybean. Soybean varieties Huipizhiheidou and Wuzhaiheidou were distributed in China and exhibited broad spectrums of resistance to various SCN races. In this study, these two resistant varieties, combined with standard susceptible varieties (Lee and Essex), were utilized to identify the differentially expressed transcripts after infection with SCN race 4 between resistant and susceptible reactions by using the Affymetrix Soybean Genome GeneChip. Comparative analyses indicated that 21 common genes changed significantly in the resistant group, of which 16 increased and 5 decreased. However, 12 common genes changed significantly in the susceptible group, of which 9 increased and 3 decreased. Additionally, 27 genes were found in common between resistant and susceptible reactions. The 21 significantly changed genes in resistant reaction were associated with disease and defense, cell structure, transcription, metabolism, and signal transduction. The fold induction of 4 from the 21 genes was confirmed by quantitative RT-PCR (qRT-PCR) analysis. Moreover, the gene ontology (GO) enrichment analyses demonstrated the serine family amino acid metabolic process and arginine metabolic process may play important roles in SCN resistance. This study provided a new insight on the genetic basis of soybean resistance to SCN race 4, and the identified resistant or resistant-related genes are potentially useful for SCN-resistance breeding in soybean.

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17β-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved.

Is the deep-sea crab Chaceon affinis able to induce a thermal stress response?

Fluctuations in the stress level of an organism are expressed in behavioural and molecular changes that can affect its ecology and survival. Our knowledge of thermal adaptations in deep-sea organisms is very limited, and this study investigates the critical thermal maximum (CTmax) and the heat-shock response (HSR) in the deep-sea crab Chaceon affinis commonly found in waters of the North East Atlantic. A mild but significant HSR in C. affinis was noted and one of the lowest CTmax known amongst Crustacea was revealed (27.5°C at 0.1MPa; 28.5°C at 10MPa). The thermal sensitivity of this species appears to be reduced at in situ pressure (10MPa), given the slightly higher CTmax and the significant 3-fold induction of stress genes hsp70 form 1 and hsp70 form 2. Although C. affinis deep-sea habitat is characterized by overall low temperature this species appears to have retained its ability to induce a HSR. This capability may be linked with C. affinis' occasional exploitation of warmer and thermally instable hydrothermal vent fields, where it has been found foraging for food.

Abstract 18576: Hypoxia Induces Phenotypic Switching of Human Pericytes Into Smooth Muscle Cells That is Associated With Changes in Pericyte-specific Non-coding RNA Expression

Background: Pericytes are pivotal for endothelial barrier integrity, angiogenesis, organ function and wound healing. While endothelial hypoxic reponses are well explored, knowledge on the effects of hypoxia on human pericytes (hPC) is sparse. Non-coding RNAs (ncRNAs) control gene expression by various mechanisms, but their functions in hPC remain unknown. We hypothesized that hypoxia influences the hPC-specific transcriptome and hPC phenotype. Methods: hPC were validated by analyzing established pericyte expression markers via qRT-PCR and immunofluorescence (IF). Subsequently, hPC and HUVEC were subjected towards hypoxia (24h 1%O 2 ), and next generation RNA deep sequencing (RNAseq) was performed. hPC viability following hypoxia was analyzed by CalceinRed assay. Results: hPC expressed specific pericyte markers (Desmin, NG2, alphaSMA, PDGFRb) on mRNA and protein level. Cell viability of hPC was not affected by hypoxia. RNAseq revealed that 109 coding genes are significantly regulated in both HUVEC and hPC, whereas 520 coding genes were significantly regulated specifically in hPC (P&lt;0.05, n=3). Interestingly, we identified a number of smooth muscle cell (SMC)-related genes that were increased in hypoxic hPC. The increase of the prototypic SMC marker alphaSMA was confirmed by qRT-PCR (2.23±1.18 fold induction) and IF. We observed a significant regulation of 6 ncRNAs, which were commonly regulated in HUVEC and in hPC, including the known hypoxia-induced miR-210 cluster (HUVEC: 7.86±0.09; hPC: 7.1±0.72 fold induction). Further 36 hPC-specific ncRNAs were identified, among them 25 long intergenic ncRNAs (lincRNAs) and 10 antisense RNAs (P&lt;0.05,n=3). Notably, lincRNA H19, that is known to be important for muscle cell differentiation, was strongly upregulated in hypoxic hPC (4.24±1.1 fold induction, P&lt;0.001,n=3). Conclusion: Hypoxia triggers hPC differentiation towards a SMC phenotype that is associated with a significant change in hPC-specific ncRNA expression without affecting hPC viability. The functions of the newly identified ncRNAs are currently investigated. Our findings may be relevant to understand the role of pericytes in persistent vascular constriction and vessel destabilization in stroke or myocardial infarction.

Ex vivo response to histone deacetylase (HDAC) inhibitors of the HIV long terminal repeat (LTR) derived from HIV-infected patients on antiretroviral therapy.

Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.

Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant

Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA+CpG ODN showed higher levels (p<0.05) of antibody titer, T cell proliferation, and percentages of CD4+ and CD8+ T cell in peripheral blood mononuclear cells (PBMCs). The relative fold inductions of mRNA expression of Th1-type (IFN-γ and IL-12), and Th2-type (IL-6) cytokines in PBMCs isolated from ducks immunized with rOmpA+CpG ODN were significantly higher than those of the rOmpA group. Homologous challenge result showed that the rOmpA+CpG ODN vaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA+CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer.

Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.

Tissue engineering (TE) strategies aim at imitating the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation, and differentiation. Based on the idea of combining a fully degradable polymer [poly(ɛ-caprolactone)] with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3±0.2) and alkaline phosphatase (ALP) activity (p<0.001) were increased compared with the control C2C12 cells. Three-dimensional osteogenic differentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation toward the myogenic or adipogenic lineage showed expression of specific markers in 3D, at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (p<0.001) as well as myoD (p<0.001) was found in C2C12 cells. The adipogenic differentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4 (FABP4) (p=0.008), and adiponectin (p=0.045) in 3D compared with 2D culture. Leptin levels in the supernatant of the ASC cultures were elevated in the 3D cultures in both donors at day 14 and 21. In conclusion, the thermoresponsive scaffold was found suitable for 3D in vitro differentiation toward soft tissue.

Prostate cancer characteristics associated with response to pre-receptor targeting of the androgen axis.

BackgroundFactors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.MethodsWe characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy.ResultsIn LuCaP35 tumors (intra-tumoral T:DHT ratio 2∶1) dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone (337 vs.152 days, HR 2.8, p = 0.0015). In LuCaP96 tumors (T:DHT 10∶1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels (5-fold higher SRD5A1, 41 fold higher, 99-fold higher RL-HSD, p<0.0001 for both), reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes (6.9-fold higher AKR1C2, 3000-fold higher UGT2B15, p = 0.002 and p<0.0001 respectively), persistent suppression of intra-tumoral DHT, and 6–8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts.ConclusionsIntrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models.

RNA-seq analysis reveals significant effects of EGFR signalling on the secretome of mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) contribute to breast cancer progression by releasing soluble factors that sustain tumor progression. MSCs express functional epidermal growth factor receptor (EGFR) and breast cancer cells secrete EGFR-ligands including transforming growth factor-α (TGFα). Using RNA-sequencing, we analysed the whole transcriptome of MSCs stimulated with TGFα. We identified 1,640 highly differentially regulated genes: 967 genes up-regulated with Fold Induction (FI) ≥ 1.50 and 673 genes down-regulated with FI ≤ 0.50. When highly regulated genes were categorized according to GO molecular function classification and KEGG pathways analysis, a large number of genes coding for potentially secreted proteins or surface receptors resulted enriched following TGFα treatment, including VEGFA, IL6, EREG, HB-EGF, LIF, NGF, NRG1, CCL19, CCL2, CCL25 and CXCL3. Secretion of corresponding proteins was confirmed for selected factors. Finally, we identified 4,377 and 4,262 alternatively spliced genes in untreated and TGFα-treated MSCs, respectively. Among these, an unannotated splice variant of VEGFA coding for a secreted VEGF protein of 172 amino acids (VEGFA(172)), was found only in MSCs stimulated with TGFα. These findings suggest that EGFR activation in MSCs leads to a significant change in the expression of a wide array of genes coding for secreted proteins that can significantly enhance tumor progression.

Abstract 3363: Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3)

Abstract In human colon cancer cells, glycolytic activity shows a positive correlation with proliferation. PFKFB3 is an important enzyme regulating glycolysis in many tumor cells. We have studied the action of an established inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation in four colon cancer cell lines (Caco-2, HCT116, HT29 and SW1116). Glucose uptake and cell proliferation were inhibited by 3PO and butyrate and additive effects were seen with 10 µM 3PO and 1 mM butyrate. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The large induction of alkaline phosphatase activity by butyrate seen in most colon cancer cell lines was not observed with 3PO. Similar contrasting effects on induction of alkaline phosphatase activity were seen with rat Morris hepatoma 7777 cells where 2-3 fold induction was seen with 1 µM vorinostat or 1 mM butyrate but no significant effect was seen with 3PO in the range of 5-30 µM. The biguanides, metformin and phenformin, increase glycolysis but inhibit proliferation of colon cancer cells. After combination treatment with 3PO, glycolysis was inhibited, as judged by glucose uptake and acidification of the medium, and additive inhibitory effects on cell proliferation could be observed. For example, after a 72 hour incubation of Caco-2 cells with 10 µM 3PO, 25 µM phenformin or 1 mM metformin, as single agents, proliferation relative to controls was decreased 20%, 17% and 34%, respectively. For combinations of 3PO and phenformin or 3PO and metformin the decreases relative to controls were 48% and 58%, respectively. In conclusion, 3PO was an inhibitor of proliferation of colon cancer cells, especially the rapidly growing HCT116 and HT29 cell lines. 3PO may be a useful agent in combination with other drugs that inhibit colon cancer cell proliferation. Citation Format: Michael A. Lea, Raymond Geherty, Rachelle David, Charles desBordes. Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2014-3363

Abstract 149: TF-FVIIa activates VEGF-A secretion in ovarian epithelial cancer cell line via a PAR-2-dependent mechanism

Abstract Background and Objective: Venous thromboembolism (VTE) is intrinsic to the biology of EOC. Progression of malignancy is associated with increased tissue factor (TF) expression. Formation of the TF-factor VIIa (FVIIa) complex triggers local thrombin generation, with thrombin activating protease activated receptor-1 (PAR-1). The TF-FVIIa complex also triggers intracellular signaling via phosphorylation of the TF C-terminus and activation of PAR-2. Although PAR-2 analysis in ovarian cancer has been limited, PAR-2 activation in renal cell carcinoma is associated with MAPK signaling pathway activation and induction of growth factor release. We hypothesize that TF-FVIIa dependent activation of PAR-2 on EOC cells up-regulates expression of angiogenic mediators (VEGF-A) that modify the peritoneal microenvironment, enhancing vascular leakage and ascites formation. Methods: TF antigen expression in EOC cell lines (CaOV-3, SKOV-3 &amp; OvCAR-3) was determined by western blot (WB) analysis. TF-bearing microparticles were isolated from conditioned media by differential centrifugation (20,800 g) and TF coagulant activity was detected by factor X activation. VEGF-A levels in conditioned media were determined by ELISA. Proliferation of the SKOV-3 and OVCAR-3 cell lines in response to thrombin (PAR-1 agonist) was determined via direct cell count. Results: WB analysis of EOC cell line lysates demonstrated PAR-2 expression (CaOV-3&amp;gt;OvCAR-3&amp;gt;&amp;gt;SKOV-3), and exposure of SKOV-3 and OVCAR-3 cells to 50 nM FVIIa resulted in 3-fold induction of VEGF-A release over 48 hr relative to untreated cells (p&amp;lt;0.05). TF-FVIIa complex was a more potent inducer of VEGF-A when compared to the PAR-2 peptide agonist (SKIGKL-NH2). Pre-incubation with the PAR-2 antagonist ENMD-1068 reduced VEGF-A to basal levels in the presence of TF-FVIIa; further supporting PAR-2 dependent induction of VEGF-A. Pan-expression of PAR-1 was observed in EOC cell lines by WB. Thrombin (0.1-1U/mL), an established PAR-1 agonist, induced dose-dependent proliferation of SKOV-3 and OVCAR-3 cell lines under serum free conditions (p&amp;lt;0.001). Pre-incubation with the PAR-1 antagonist vorapaxar reduced proliferation to basal levels in the presence of thrombin. Conclusion: TF expression and coagulant activity is up regulated in EOC. FVIIa triggers a PAR-2 dependent induction of VEGF-A release from EOC cell lines. Similarly, the PAR-1 agonist thrombin triggered a significant increase in EOC cell proliferation. These results suggest that interaction of EOC with the coagulation system in the tumor microenvironment may enhance both PAR-1 dependent cellular proliferation and PAR-2 dependent release of VEGF, a potent regulator of angiogenesis. Thus, PAR-1 and PAR-2 may represent pharmacologically relevant drug targets to block ovarian cancer progression via inhibition of growth and angiogenesis. Citation Format: Alice Chanakira, John P. Sheehan. TF-FVIIa activates VEGF-A secretion in ovarian epithelial cancer cell line via a PAR-2-dependent mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 149. doi:10.1158/1538-7445.AM2014-149

Pharmacological relevance of endoxifen in a laboratory simulation of breast cancer in postmenopausal patients.

Tamoxifen is metabolically activated via a CYP2D6 enzyme system to the more potent hydroxylated derivatives 4-hydroxytamoxifen and endoxifen. This study addresses the pharmacological importance of endoxifen by simulating clinical scenarios in vitro. Clinical levels of tamoxifen metabolites in postmenopausal breast cancer patients previously genotyped for CYP2D6 were used in vitro along with clinical estrogen levels (estrone and estradiol) in postmenopausal patients determined in previous studies. The biological effects on cell growth were evaluated in a panel of estrogen receptor-positive breast cancer cell lines via cell proliferation assays and real-time polymerase chain reaction (PCR). Data were analyzed with one- and two-way analysis of variance and Student's t test. All statistical tests were two-sided. Postmenopausal levels of estrogen-induced proliferation of all test breast cancer cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 11 ± 1.74, P < .001; T47D = 7.52 ± 0.72, P < .001; BT474 = 1.75 ± 0.23, P < .001; ZR-75-1 = 5.5 ± 1.95, P = .001. Tamoxifen and primary metabolites completely inhibited cell growth regardless of the CYP2D6 genotype in all cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 1.57 ± 0.38, P = .54; T47D = 1.17 ± 0.23, P = .79; BT474 = 0.96 ± 0.2, P = .98; ZR-75-1 = 0.86 ± 0.67, P = .99). Interestingly, tamoxifen and its primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting.

Protein Modifications Regulate the Role of 14-3-3γ Adaptor Protein in cAMP-induced Steroidogenesis in MA-10 Leydig Cells

The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser(58) phosphorylation and 14-3-3γ Lys(49) acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser(58) or Lys(49). Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser(58) phosphorylation and Lys(49) acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser(58) phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys(49) acetylation is important for the cAMP-dependent induction of these interactions.

Differential regulation of TGF-β-induced, ALK-5-mediated VEGF release by SMAD2/3 versus SMAD1/5/8 signaling in glioblastoma.

The transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) pathways have a major role in the pathogenesis of glioblastoma, notably immunosuppression, migration, and angiogenesis, but their interactions have remained poorly understood. We characterized TGF-β pathway activity in 9 long-term glioma cell lines (LTCs) and 4 glioma-initiating cell lines (GICs) in relation to constitutive and exogenous TGF-β-induced VEGF release. Results were validated using The Cancer Genome Atlas transcriptomics data. Glioma cells exhibit heterogeneous patterns of constitutive TGF-β pathway activation reflected by phosphorylation not only of SMAD2 and SMAD3 but also of SMAD1/5/8. Constitutive TGF-β pathway activity depends on the type I TGF-β receptor, ALK-5, and accounts for up to 69% of constitutive VEGF release, which is positively regulated by SMAD2/3 and negatively regulated by SMAD1/5/8 signaling in a cell line-specific manner. Exogenous TGF-β induces VEGF release in most cell lines in a SMAD- and ALK-5-dependent manner. There is no correlation between the fold induction of VEGF secretion induced by TGF-β compared with hypoxia. The role of SMAD5 signaling is highly context and cell-line dependent with a VEGF inhibitory effect at low TGF-β and pSMAD2 levels and a stimulatory effect when TGF-β is abundant. TGF-β regulates VEGF release by glioma cells in an ALK-5-dependent manner involving SMAD2, SMAD3, and SMAD1/5/8 signaling. This crosstalk between the TGF-β and VEGF pathways may open up new avenues of biomarker-driven exploratory clinical trials focusing on the microenvironment in glioblastoma.