Targeting Ornithine Decarboxylase Research Articles

Drug repositioning to discover novel ornithine decarboxylase inhibitors against visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by Leishmania donovani (Ld), and most cases occur in Brazil, East Africa, and India. The treatment for VL is limited and has many adverse effects. The development of safer and more efficacious drugs is urgently needed. Drug repurposing is one of the best processes to repurpose existing drugs. Ornithine decarboxylase (ODC) is an important target against L. donovani in the polyamine biosynthesis pathway. In this study, we have modeled the 3D structure of ODC and performed high-throughput virtual screening of 8630 ZINC database ligands against Leishmania donovani ornithine decarboxylase (Ld ODC), selecting 45 ligands based on their high binding score. It is further validated through molecular docking simulation and the selection of the top two lead molecules (ceftaroline fosamil and rimegepant) for Molecular Dynamics (MD) simulation, Density functional theory (DFT), and molecular mechanics generalized born surface area (MMGBSA) analysis. The results showed that the binding affinities of ceftaroline fosamil, and rimegepant are, respectively, -10.719 and 10.159 kcal/mol. The docking complexes of the two lead compounds, ceftaroline fosamil, and rimegepant, with the target ODC, were found stable during molecular dynamics simulations. Furthermore, the analysis of MMGBSA revealed that these compounds had a high binding free energy. The DFT analysis showed that the top lead molecules were more reactive than the standard drug (pentamidine). In-silico findings demonstrated that ceftaroline fosamil, and rimegepant might be recognized as potent antagonists against ODC for the treatment of VL.

Heterologous expression and activity verification of ornithine decarboxylase from a wild strain of Shewanella xiamenensis.

Shewanella xiamenensis is widely found in spoilage fish, shrimp and other seafoods. Under suitable conditions, ornithine can be synthesized into putrescine, which may spoil food or endanger health. Our research used a wild strain of Shewanella xiamenensis isolated from "Yi Lu Xian" salted fish (a salting method for sea bass) as a research object. According to the database of National Center of Biotechnology Information (NCBI), the target ornithine decarboxylase (ODC) gene SpeF was successfully amplified using the wild strain of Shewanella xiamenensis as the template. Sequencing alignment showed that the SpeF of the wild strain had more than 98% homology compared with the standard strain. The amino acid substitution occurred in the residues of 343, 618, 705, and 708 in the wild strain. After optimizing the expression conditions, a heterologous expression system of ODC was constructed to achieve a high yield of expression. The amount of 253.38 mg of ODC per liter of LB broth was finally expressed. High performance liquid chromatography (HPLC) and subsequent ODC activity verification experiments showed that hetero-expressed ODC showed a certain enzyme activity for about 11.91 ± 0.38 U/mg. Our study gives a new way to the development of a low-cost and high-yield strategy to produce ODC, providing experimental materials for further research and elimination of putrescine in food hazards.

Structure based design of inhibitory peptides targeting ornithine decarboxylase dimeric interface and in vitro validation in human retinoblastoma Y79 cells

Polyamine synthesis in human cells is initiated by catalytic action of Ornithine decarboxylase (ODC) on Ornithine. Elevated levels of polyamines are manifested proliferating cancer cells and are found to promote tumour cell adhesion. Di-flouro methyl orninthine is a known inhibitor of ODC, however its usage is limited due its low affinity quick clearance and incompetent cellular uptake, thus posing a need for potential inhibitors. Currently, peptides are substituting drugs, as these are highly selective, specific and potent. Hence, in this study, the interacting interfaces of native homodimeric form of ODC and its heterodimer with Antizyme were probed to design inhibitory peptides targeting ODC. The designed peptides were validated for structural fitness by extensive molecular dynamics simulations and Circular dichroism studies. Finally, these peptides were validated in Y79 retinoblastoma cells for impact on ODC activity, cytotoxicity cell cycle and cell adhesion. On collective analysis, Peptide3 (Pep 3) and Peptide4 (Pep 4) were found to be potentially targeting ODC, as these peptides showed significant decrease in intracellular polyamine levels, cell adhesion and cell cycle perturbation in Y79 cells. Thus, Pep 3 and Pep 4 shall be favourably considered as therapeutic agents for targeting ODC mediated proliferation in retinoblastoma. Communicated by Ramaswamy H. Sarma

Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ95–228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the α1 helix, β5 and β6 strands, and connecting loop between β6 and α2 (residues 142–178), which is the posterior part of AZ95–228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ; amino acid residues within the anterior part of AZ95–228 (residues 120–145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

Abstract 1788: Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer

Abstract Precancerous lesion of esophageal squamous cell cancer (ESCC) owns the characteristic of bidirectional instability, which can develop to cancer or reverse to normal epithelium by appropriate interference. Therefore, the feature of precancerous lesion provides possibility for ESCC chemoprevention. In the current study, we found that ornithine decarboxylase (ODC) protein level is higher in esophageal precancerous lesions compared with normal esophageal epithelium. Knocking down ODC suppresses cell proliferation and clone formation of ESCC. Moreover, difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreases N-nitrosomethylbenzylamine (NMBA)-induced rats’ esophageal precancerous lesions. Further molecular study indicated that ODC down-regulates p38, ERK in the MAP kinase pathway and AKT/mTOR/p70S6K pathway, which are involved in the carcinogenesis process. Overall, our results indicate that ODC may be a potential target for ESCC chemoprevention. Citation Format: Yifei Xie, Qiong Wu, Jimin Zhao, Kangdong Liu. Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1788.

Ornithine Decarboxylase in Macrophages Exacerbates Colitis and Promotes Colitis-Associated Colon Carcinogenesis by Impairing M1 Immune Responses.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis and restricts M1 macrophage activation in gastrointestinal (GI) infections. However, the role of macrophage ODC in colonic epithelial-driven inflammation is unknown. Here, we investigate cell-specific effects of ODC in colitis and colitis-associated carcinogenesis (CAC). Human colonic macrophages expressed increased ODC levels in active ulcerative colitis and Crohn's disease, colitis-associated dysplasia, and CAC. Mice lacking Odc in myeloid cells (OdcΔmye mice) that were treated with dextran sulfate sodium (DSS) exhibited improved survival, body weight, and colon length and reduced histologic injury versus control mice. In contrast, GI epithelial-specific Odc knockout had no effect on clinical parameters. Despite reduced histologic damage, colitis tissues of OdcΔmye mice had increased levels of multiple proinflammatory cytokines and chemokines and enhanced expression of M1, but not M2 markers. In the azoxymethane-DSS model of CAC, OdcΔmye mice had reduced tumor number, burden, and high-grade dysplasia. Tumors from OdcΔmye mice had increased M1, but not M2 macrophages. Increased levels of histone 3, lysine 9 acetylation, a marker of open chromatin, were manifest in tumor macrophages of OdcΔmye mice, consistent with our findings that macrophage ODC affects histone modifications that upregulate M1 gene transcription during GI infections. These findings support the concept that macrophage ODC augments epithelial injury-associated colitis and CAC by impairing the M1 responses that stimulate epithelial repair, antimicrobial defense, and antitumoral immunity. They also suggest that macrophage ODC is an important target for colon cancer chemoprevention.Significance: Ornithine decarboxylase contributes to the pathogenesis of colitis and associated carcinogenesis by impairing M1 macrophage responses needed for antitumoral immunity; targeting ODC in macrophages may represent a new strategy for chemoprevention. Cancer Res; 78(15); 4303-15. ©2018 AACR.

Microarray analysis of obese women with polycystic ovary syndrome for key gene screening, key pathway identification and drug prediction

PurposeThis study aimed to screen key genes and pathways involved in obese polycystic ovary syndrome (PCOS), and predict drugs for treatment of obese PCOS via bioinformatics approaches. MethodsMicroarray dataset GSE10946 were downloaded from the Gene Expression Omnibus database, including 7 cumulus cell samples from obese PCOS patients and 6 lean control samples. Differentially expressed genes (DEGs) between obese PCOS and controls were obtained using Bayesian test after data preprocessing, followed by functional enrichment analyses for DEGs. Besides, protein-protein interaction (PPI) network and sub-network analyses were performed. Furthermore, drug prediction was carried out based on the DEGs. ResultsA total of 793 DEGs were identified in PCOS compared with control, including 352 up-regulated and 441 down-regulated DEGs. Specifically, upregulated RNA polymerase I subunit B (POLR1B), DNA polymerase epsilon 3, accessory subunit (POLE3), and DNA polymerase delta 3, accessory subunit (POLD3) were enriched in pathway of pyrimidine metabolism associated with obesity and PCOS, and 5-hydroxytryptamine receptor 2C (HTR2C) was enriched calcium signaling pathway. Additionally, 10 significant potential drugs, such as spironolactone targeting androgen receptor (AR), trimipramine targeting adrenoceptor beta 2 (ADRB2), and L-ornithine targeting ornithine decarboxylase antizyme 3 (OAZ3), were obtained. ConclusionsIn conclusion, POLR1B, POLE3, POLD3, and HTR2C might play important roles in obese PCOS via involvement of pyrimidine metabolism and calcium signaling pathway. Moreover, AR, ADRB2, and OAZ3 might be targets of spironolactone, trimipramine, and L-ornithine in the treatment of obese PCOS.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively. Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Virtual screening of natural inhibitors targeting ornithine decarboxylase with pharmacophore scaffolding of DFMO and validation by molecular dynamics simulation studies

Ornithine decarboxylase (ODC) is an enzyme that initiates polyamine synthesis in human. Polyamines play key roles in cell–cell adhesion, cell motility and cell cycle regulation. Higher synthesis of polyamines also occurs in rapidly proliferating cancer cells are mediated by ODC. As per earlier studies, di-flouro-methyl-orninthine (DFMO) is a proven efficient inhibitor ODC targeting the catalytic activity, however, its usage is limited due to side effects. Targeting ODC is considered as a potential therapeutic modality in the treatment of cancer. In this study, it is attempted to use DFMO scaffold to build a ligand-based pharmocophore query using MOE to screen similar active compounds from Universal Natural Products Database with better ADMET properties. The identified compounds were virtually screened against the active cavity of ODC using Glide. Further, potential natural hits targeting ODC were shortlisted based on Molecular Mechanics/Generalized-Born/Surface Area (MM-GBSA) score. Finally, molecular dynamics simulations were performed for the natural molecule hit and DFMO in complex with ODC using Desmond. Among the hits shortlisted, 2-amino-5, 9, 13, 17-tetramethyloctadeca-8, 16-diene-1, 3, 14-triol (UNPD208110) was found to be highly potential, as it showed a higher binding affinity in terms of interactions with key active cavity residues, and also showed better ADMET property, HUMO–LUMO gap energy and more stable complex formation with ODC compared to DFMO. Hence, the proposed molecule (UNPD208110) shall be favourably considered as a potential natural inhibitor targeting ODC-mediated disease conditions.

Inhibition of polyamine biosynthesis for toxicity control in Serratia marcescens strain WW4 by targeting ornithine decarboxylase: a structure-based virtual screening study

Inhibition of polyamine biosynthesis for toxicity control in Serratia marcescens strain WW4 by targeting ornithine decarboxylase: a structure-based virtual screening study

Inhibition of polyamine biosynthesis for toxicity control in Serratia marcescens strain WW4 by targeting ornithine decarboxylase: a structure-based virtual screening study

Ornithine decarboxylase (ODC) enzyme, catalyses the decarboxylation of ornithine to form spermidine which is a committed step in the biosynthesis of polyamines. Here, in the present work, structure of ODC was modelled and studied for its active site. The stability of modelled structure was revalidated by the molecular dynamics simulation at 50 nano second time scale. 142 Natural products of Indofine Herbal Ingredient library from ZINC database were screened using Autodock Vina for the identification of potential leading herbal inhibitors. The results obtained from docking showed that Conessine is best inhibiting candidate with docking affinity of –9.7 Kcal/mol. Conessine is an alkaloid which proves its immense importance as metabolites. Thus, polyamine being harmful when synthesised in excess are necessary to be controlled at their genesis. Therefore, conessine might be a potential inhibitor for toxicity control in plant growth promoting rhizobacteria.

Insights on ornithine decarboxylase silencing as a potential strategy for targeting retinoblastoma

Ornithine Decarboxylase (ODC) is a key enzyme involved in polyamine synthesis and is reported to be up regulated in several cancers. However, the effect of ODC gene silencing in retinoblastoma is to be understood for utilization in therapeutic applications. Hence, in this study, a novel siRNA (small interference RNA) targeting ODC was designed and validated in Human Y79 retinoblastoma cells for its effects on intracellular polyamine levels, Matrix Metalloproteinase 2 & 9 activity and Cell cycle. The designed siRNA showed efficient silencing of ODC mRNA expression and protein levels in Y79 cells. It also showed significant reduction of intracellular polyamine levels and altered levels of oncogenic LIN28b expression. By this study, a regulatory loop is proposed, wherein, ODC silencing in Y79 cells to result in decreased polyamine levels, thereby, leading to altered protein levels of Lin28b, MMP-2 and MMP-9, which falls in line with earlier studies in neuroblastoma. Thus, by this study, we propose ODC silencing as a prospective strategy for targeting retinoblastoma.

Antiproliferative and antimicrobial efficacy of the compounds isolated from the roots of Oenothera biennis L.

Oenothera biennis L., commonly known as evening primrose, harbours the flavonoids, steroids, tannins, fatty acids and terpenoids responsible for a diverse range of biological activity, such as antitumour, anti-arthritic and anti-inflammatory effects. In addition to the previous reports from aerial parts of this plant, studies related to antiproliferative or antimicrobial activity from the roots are warranted. To investigate antiproliferative and antimicrobial activity of compounds/mixture (1-8) isolated and characterized from the roots of O.biennis L. A possible mechanism of antiproliferative activity was also studied by targeting ornithine decarboxylase (ODC) and cathepsin D (CATD). Antiproliferative efficacy of the compounds/mixture was examined in selected cancer cell lines along with their probable mechanism of action. The antimicrobial activity was also studied against selected microbes (bacteria and fungi). Antiproliferative potential was evaluated by MTT assay against selected cell lines. The mechanism of action was studied spectrophotometrically by targeting ODC and CATD using both an in-vitro and an in-silico approach. The antimicrobial efficiency was analysed using the disc diffusion and broth dilution methods. Oenotheralanosterol B (3) and the mixture of oenotheralanosterol A and oenotheralanosterol B (4) exhibited antiproliferative activity against breast, hepatic, prostate and leukaemia cancer cell lines as well as in mouse macrophages (IC50 8.35-49.69μg/ml). Oenotheralanosterol B (3) and the mixture of oenotheralanosterol A and oenotheralanosterol B (4) displayed a strong molecular interaction with succinate dehydrogenase (binding energy -6.23 and -6.84kcal/mol and Ki 27.03 and 9.6μm, respectively). Oenotheralanosterol A (1), oenotheralanosterol B (3) and mixture of oenotheralanosterol A and oenotheralanosterol B (4) potently inhibited the ODC activity with IC50 ranging from 4.65±0.35 to 19.06±4.16μg/ml and also showed a strong interaction with ODC (BE -4.17 to -4.46kcal/mol). Oenotheralanosterol A (1), cetoleilyl diglucoside (2), oenotheralanosterol B (3), dihydroxyprenylxanthone acetylated (6) and dihydroxyprenylxanthone (7) inhibited CATD activity (IC50 3.95±0.49 to 24.35±2.89μg/ml). The in-silico molecular interaction analysis of compounds with CATD revealed the non-specific interaction. A moderate antimicrobial activity was observed against selected microbes with a growth inhibition ranging from 6 to 14mm and minimum inhibitory concentration between 125 and 500μg/ml. Oenotheralanosterol B (3) and dihydroxyprenylxanthone acetylated (6) exhibited better antimicrobial activity with an MIC range from 62.50 to 500μg/ml. Oenotheralanosterol B (3) exhibited stronger antiproliferative and antimicrobial potential with respect to the other compounds tested, whereas oenotheralanosterol A (1) was a potent inhibitor of ODC and CATD. Hence, it is suggested that these in-vitro findings could be studied further invivo for biological activity, safety evaluation and derivatization to enhance potency and efficacy.

Targeting ornithine decarboxylase (ODC) inhibits esophageal squamous cell carcinoma progression

To explore the function of ornithine decarboxylase in esophageal squamous cell carcinoma progression and test the effectiveness of anti-ornithine decarboxylase therapy for esophageal squamous cell carcinoma. In this study, we examined the expression pattern of ornithine decarboxylase in esophageal squamous cell carcinoma cell lines and tissues using immunohistochemistry and Western blot analysis. Then we investigated the function of ornithine decarboxylase in ESCC cells by using shRNA and an irreversible inhibitor of ornithine decarboxylase, difluoromethylornithine. To gather more supporting pre-clinical data, a human esophageal squamous cell carcinoma patient-derived xenograft mouse model (C.B-17 severe combined immunodeficient mice) was used to determine the antitumor effects of difluoromethylornithine in vivo. Our data showed that the expression of the ornithine decarboxylase protein is increased in esophageal squamous cell carcinoma tissues compared with esophagitis or normal adjacent tissues. Polyamine depletion by ODC shRNA not only arrests esophageal squamous cell carcinoma cells in the G2/M phase, but also induces apoptosis, which further suppresses esophageal squamous cell carcinoma cell tumorigenesis. Difluoromethylornithine treatment decreases proliferation and also induces apoptosis of esophageal squamous cell carcinoma cells and implanted tumors, resulting in significant reduction in the size and weight of tumors. The results of this study indicate that ornithine decarboxylase is a promising target for esophageal squamous cell carcinoma therapy and difluoromethylornithine warrants further study in clinical trials to test its effectiveness against esophageal squamous cell carcinoma.

Targeting Ornithine Decarboxylase by α-Difluoromethylornithine Inhibits Tumor Growth by Impairing Myeloid-Derived Suppressor Cells.

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.

Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma.

LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.

Effects of ornithine decarboxylase antizyme 1 on the proliferation and differentiation of human oral cancer cells.

Ornithine decarboxylase antizyme 1 (OAZ1) is an antizyme targeting ornithine decarboxylase for degradation, subsequently inhibiting polyamine production to prevent cell proliferation. OAZ1 is also involved in other major cellular events, including differentiation and apoptosis. Recent studies have shown that OAZ1 has tumor suppressor activities and its effects on cell proliferation and differentiation have been reported in several cancer cell lines. To explore the role of OAZ1 in human oral cancer, the effects of OAZ1 were studied on the proliferation and differentiation of human malignant oral cancer cell line, SCC15. MTT assay and flow cytometry analysis showed that stable OAZ1 expression in SCC15 significantly inhibited cell proliferation (P<0.001) and induced G0/G1 arrest with the G1‑phase cells increased from 55.83 to 65.26%. Morphological observation revealed the increased formation of epithelial islands. Further results from quantitative reverse transcription‑polymerase chain reaction and western blot analysis proved the upregulation of several terminal differentiation marker genes (K10, FLG and LOR) in OAZ1‑expressed SCC15 cells. To elucidate the possible mechanism of LOR upregulation by OAZ1, further experiments were performed and it was found that the OAZ1 expression inhibited Smad nuclear interacting protein 1 (SNIP1) at the protein level and RNA interference of SNIP1 in SCC15 cells, which increased the expression of LOR. These results show that OAZ1 simultaneously inhibits the proliferation and induces the differentiation of oral cancer cells in humans. The effects on cellular differentiation depend partly on the degradation of SNIP1.

51 BVES Orchestrates Pr61α-PP2A Mediated C-Myc Degradation

Background: Blood vessel epicardial substance (BVES) is a tight-junction associated protein that regulates epithelial-to-mesenchymal transition (EMT). BVES is underexpressed in ulcerative colitis (UC), and the BVES promoter is hypermethylated in colitis-associated cancer (CAC). We have recently presented that Bves mice have greater tumor burden and higher grade dysplasia in the AOM/DSS model of CAC. Here we defined the mechanism by which BVES regulates EMT and CAC. Methods: After tumor induction using AOM/DSS, intratumoral RNA was isolated from Bves and WT mice and the transcriptome defined via RNAseq. Tumors were stained by IHC for c-myc and phospho-S62-c-myc and scored using an index based on positive cells per HPF. C-myc protein analysis was conducted in HEK 293T, HCT116, and SW480 cell lines. Enteroid cultures were isolated from 4-week old mice and harvested after 72 hours for RNA and protein. Cells were treated with 100ug/ml of cycloheximide 72 hours after transfection. Polyhis-tagged ubiquitin was co-transfected with HA-c-myc with empty vector or V5-BVES. Ubiquitin was pulled down using Ni-NTA agarose and c-myc was blotted. Transepithelial resistance (TER) was measured daily for 7 days. WST-1 values were recorded daily for 3 days post-plating. Results: RNA-seq analysis using Ingenuity Pathway identified that the c-myc transcriptional network was disrupted in AOM/ DSS Bves tumors. IHC analysis of c-myc and phopsho-S62c-myc showed an increase in Bves tumors (c-myc 2.6± 0.1 vs. 1.8± 0.1 P<0.0001 and phospho-S62-c-myc 2.8± 0.2 vs. 2.1± 0.1 per tumor HPF P<0.01). Bves enteroids had 10-fold upregulation of c-myc protein and a 38-fold increase in the c-myc transcriptional target ornithine decarboxylase. Knocking down BVES in 293T, HCT116, and SW480 cells increased c-myc protein levels by 25%. Cycloheximide treatment after BVES knockdown doubled c-myc half-life. Further, transfection of BVES increased poly-ubiquitylated c-myc suggesting BVES promotes c-myc degradation. Lastly, immunopreciptation demonstrated that BVES and c-myc exist in complex. A yeast-two-hybrid screen identified PR61α, a regulatory subunit of the PP2A family of phosphatases known to promote c-myc degradation, interacts with BVES. We confirmed the BVES:PR61α interaction by directed Y2H and co-IP. PR61α knockdown blocked BVESinduced reduction of c-myc, suggesting BVES requires PR61α to promote c-myc destruction. Caco-2 cell differentiation requires post-translational downregulation of c-myc. When PR61α was knocked down, BVES was unable to increase TER in Caco-2 cells. Further, c-myc promotes proliferation, and PR61α knockdown prevented BVES-induced reduction of WST1. Conclusions: BVES requires PR61α to regulate TER and proliferation, potentially through directing the c-myc downregulation.

Abstract 2765: Targeting both ornithine decarboxylase and polyamine transport inhibits tumor growth in neuroblastoma.

Abstract Background: Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested on NB. We proposed that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501, would result in increased NB growth inhibition. Methods: Single and combination drug treatments were conducted on three NB cell lines in vitro and in a xenograft mouse model in vivo. Cell viability was measured using Calcein AM fluorescent assay. Real-time cell proliferation was measured using the xCELLigence system. Western blot analysis was used to measure cleaved and full PARP, cleaved and full caspase 3, phosphorylated Rb and MYCN levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Reverse-phase HPLC was used to measure polyamine levels in cells. Results: DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 μM in NB cell lines. Low dose combination treatment (2.5 μM AMXT-1501, 2.5 mM DFMO) synergistically inhibited cell viability and proliferation in vitro. Combination treatment decreased MYCN expression and resulted in hypophosphorylation of pRb, suggesting cell growth inhibition. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hours suggested apoptosis. The combination treatment resulted in intracellular polyamine pool depletion and decreased intracellular ATP. The combination treatment also significantly reduced tumor size in NB xenograft mice in vivo. Conclusion: Given the current lack of effective therapies for relapsed/refractory NB patients and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB. Citation Format: Ann M. Kendzicky, Ping Zhao, Katherine Samal, Lisette P. Yco, Heather McClung, Eugene W. Gerner, André S. Bachmann, Giselle L. Sholler. Targeting both ornithine decarboxylase and polyamine transport inhibits tumor growth in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2765. doi:10.1158/1538-7445.AM2013-2765

Abstract 3808: MYCN coordinately regulates the entire polyamine gene pathway to drive polyamine biosynthesis in neuroblastoma, with expression of every synthetic and catabolic polyamine pathway gene being highly prognostic of clinical outcome.

Abstract Background: Polyamines are highly regulated essential cations that are elevated in rapidly proliferating tissues. We have previously shown that the MYC/MYCN target ornithine decarboxylase (ODC1), rate-limiting for polyamine biosynthesis, is a therapeutic target for neuroblastoma (Hogarty et al, Cancer Res 2008; 68: 9735). We have also shown that targeting multiple steps in the polyamine pathway enhances therapeutic efficacy in complementary pre-clinical models (Haber et al, Adv Neurobl Res, 2012, OR 88). An international Phase I trial for refractory neuroblastoma, coordinated by the New Approaches to Neuroblastoma Treatment (NANT) consortium USA, will open shortly using ODC1 inhibition by high-dose difluoromethylornithine and SAT1 induction by celecoxib. We have now examined the prognostic impact of all polyamine pathway genes in a large cohort of neuroblastoma tumors. Methods: Gene-expression profiles of 650 primary untreated neuroblastomas were analyzed for all polyamine pathway genes, and related to clinical outcome. Gene expression was evaluated by Real-Time PCR (RT-PCR) and correlated with MYCN levels in neuroblastoma cell lines. Activation and repression of polyamine genes by MYCN were analyzed by chromatin immunoprecipitation (ChIP). Polyamine gene promoters were cloned into luciferase reporter vectors and tested as a function of MYCN expression. Results: Expression of all polyamine pathway genes was highly prognostic of neuroblastoma outcome. High levels of each synthetic gene and low levels of each catabolic gene predicted poor outcome. Multivariate analysis showed 6/11 genes retained independent prognostic significance following adjustment for MYCN, age and stage. In neuroblastoma cell lines, in the presence or absence of MYCN expression, RT-PCR analysis demonstrated a correlation of high MYCN with high biosynthetic polyamine gene expression, but an inverse correlation of high MYCN with low catabolic polyamine gene expression. ChIP assays confirmed that MYCN associates with biosynthetic gene promoters by binding to E-box sites in order to activate transcription. In contrast, MYCN binds the promoters of the catabolic genes in proximity to the transcription start sites by interacting with the Sp1 protein in order to repress transcription of these genes. Direct activation by MYCN of biosynthetic genes and down-regulation by MYCN of catabolic genes was confirmed by luciferase activity assays. Conclusions: These results provide a remarkable demonstration of an oncogene coordinately regulating the expression of every gene in a metabolic pathway in order to drive cell proliferation. The findings highlight the importance of polyamines in neuroblastoma and identify target enzymes in addition to ODC1 for potential therapeutic intervention. Citation Format: Murray D. Norris, Laura D. Gamble, Jayne Murray, Stefania Purgato, Giorgio Milazzo, Lesley J. Ashton, Andrei Oberthuer, Wendy B. London, Mike D. Hogarty, Mattias Fischer, David Ziegler, Glenn M. Marshall, Giovanni Perini, Michelle Haber. MYCN coordinately regulates the entire polyamine gene pathway to drive polyamine biosynthesis in neuroblastoma, with expression of every synthetic and catabolic polyamine pathway gene being highly prognostic of clinical outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3808. doi:10.1158/1538-7445.AM2013-3808