Ornithine Decarboxylase Expression Research Articles

Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias

The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.

Polyamine Depletion Attenuates Isoproterenol-Induced Hypertrophy and Endoplasmic Reticulum Stress in Cardiomyocytes

Background/Aim: Polyamines (putrescine, spermidine and spermine) play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS) in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy. Methods: Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO). Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO). Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting. Results: DFMO (0.5 mM and 2 mM) treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH) and malondialdehyde (MDA) level in the culture medium. In addition, DFMO (0.5 mM) down regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM). Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

Downregulation of c-Myc mediated ODC expression after purvalanol treatment is under control of upstream MAPK signaling axis in MCF-7 breast cancer cells

Roscovitine and purvalanol are specific cyclin-dependent kinase (CDK) inhibitors, which induce apoptosis by triggering cell cycle arrest in various cancer cells such as colon, prostate, and breast cancer cells. Although the apoptotic action of roscovitine was clarified at the molecular level, the exact mechanism of purvalanol-induced apoptosis is still under investigation. The mitogen-activated protein kinase (MAPK) signaling cascade is activated by different inducers related to growth, proliferation, differentiation processes, or environmental stress factors. Recent reports showed that modulation of MAPKs might lead to regulation of c-Myc, which is a transcription factor for the polyamine (PA) biosynthesis enzyme, ornithine decarboxylase (ODC). PAs are amine-derived cationic molecules that play crucial roles in cell proliferation, growth, and differentiation. In this study, we investigated the potential role of the MAPK signaling cascade in the purvalanol-induced apoptosis mechanism by comparing the results of roscovitine in MCF-7 and MDA-MB-231 breast cancer cells. We found that CDK inhibitors decreased the cell viability in a dose- and time-dependent manner in MCF-7 and MDA-MB-231 cancer cells. Although both CDK inhibitors induced cell cycle arrest, which led to apoptosis by activating caspases and PARP cleavage in MCF-7 breast cancer cells, the apoptotic effect of purvalanol was less than that of roscovitine in MDA-MB-231 cells. Inhibition of MAPKs prevented CDK inhibitor-induced cell viability loss in both cell lines. We determined that purvalanol downregulated c-Myc and ODC expression levels, which led to sharp decrease in the PA pool in MCF-7 cells. On the contrary, purvalanol did not significantly alter c-Myc expression levels, which led to de novo biosynthesis of ODC in a time-dependent manner in MDA-MB-231 cells. Therefore, we suggest that a purvalanol-mediated resistance phenotype might be a possible outcome of c-Myc-mediated ODC expression level in MDA-MB-231 cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Hairless and the polyamine putrescine form a negative regulatory loop in the epidermis

Hairless (HR) is a nuclear protein with corepressor activity that is highly expressed in the skin and hair follicle. Mutations in Hairless lead to hair loss accompanied by the appearance of papules (atrichia with papular lesions), and similar phenotypes appear when the key polyamine enzymes ornithine decarboxylase (ODC) and spermidine/spermine N(1) -acetyltransferase (SSAT) are overexpressed. Both ODC and SSAT transgenic mice have elevated epidermal levels of putrescine, leading us to investigate the mechanistic link between putrescine and HR. We show here that HR and putrescine form a negative regulatory network, as epidermal ODC expression is elevated when HR is decreased and vice versa. We also show that the regulation of ODC by HR is dependent on the MYC superfamily of proteins, in particular MYC, MXI1 and MXD3. Furthermore, we found that elevated levels of putrescine lead to decreased HR expression, but that the SSAT-TG phenotype is distinct from that found when HR is mutated. Transcriptional microarray analysis of putrescine-treated primary human keratinocytes demonstrated differential regulation of genes involved in protein-protein interactions, nucleotide binding and transcription factor activity, suggesting that the putrescine-HR negative regulatory loop may have a large impact on epidermal homeostasis and hair follicle cycling.

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.

Interactive effects of 1, 25-dihydroxyvitamin D3 and soy protein extract (SPE) on oral cancer growth in vitro: evidence for potential functional relationships.

Background: Previous studies have found specific soy isoflavones (Genistein, Daidzein, Glycitein) demonstrate anti-tumor properties against several cancer types, including oral cancer. Few studies have evaluated whole soy extract, containing a combination of these isoflavones and other bioreactive compounds, which may function synergistically and more effectively against oral cancers. Preliminary work by this group has now demonstrated whole soy protein extract (SPE) inhibits oral cancer cell growth specifically and selectively, through independent cell-cycle and apoptotic pathways. However, more recent evidence now suggests that ingestion of vitamin D3, either in dietary foods or supplements may potentiate the activity of soy components and their anti-tumor effects.Objective: The primary goal of this study was to investigate the interactive and inter-connected effects of 1, 25-dihydroxyvitamin D3 administration with the anti-proliferative effects of whole soy protein extract (SPE) on oral cancer and normal cell lines in vitro.Methods: Three oral squamous cell carcinoma cell lines (SCC15, SCC25, and CAL27) were treated with 1, 25-dihydroxy Vitamin D3 at physiological concentrations (10-125 nmol). Cell growth was then compared with cell treatment using soy protein extract (SPE) within the normal physiologic range (0 - 10 μM/L). Interactive effects were then evaluated using co-administration of SPE and 1, 25-dihydroxy Vitamin D3. Quantitative RT-PCR was performed at various time points to determine any changes in mRNA expression for key cell cycle and apoptotic signaling pathway regulators, including p53, c-myc, ornithine decarboxylase (ODC), caspase-2, caspase-8, and bax. Results: Administration of 1, 25-dihydroxy Vitamin D3 induced distinct dose-dependent, growth-inhibitory effects in all three oral cancer cell lines examined. These inhibitory effects were comparable to the overall range of growth inhibition induced by SPE. However, the combined effects of co-administration were far greater, suggesting the presence of synergistic relationships between these components. In addition, these results indicate that either treatment alone appeared to modulate mRNA expression of oral cancer cell-cycle promoters c-myc and ODC, as well as the caspase-dependent apoptosis pathway, while only 1, 25-dihydroxy Vitamin D3 administration appeared to influence the bax pathway. Conclusion: These results suggest that co-administration with 1, 25-dihydroxy Vitamin D3 and SPE may enhance their anti-tumor effects. This study may help to explain, in part, why balanced diets rich in fruits, vegetables, and soy protein, are associated with protection against development and progression of oral cancers, although further study is needed to develop specific public health recommendations for oral cancer treatment and prevention.Key words: vitamin D, soy extract, whole soy protein, oral cancer, growth inhibition.

AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport

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 many forms of cancer including 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 in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. 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. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB 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.

101 Ornithine Decarboxylase Disrupts Host Immune Tolerance in Helicobacter pylori Infection by Attenuating Macrophage Production of TGF-β and Nitric Oxide

Background: Helicobacter pylori (Hp) infection results in a Th1/Th17 polarized inflammatory response. We have reported that expression of ornithine decarboxylase (ODC), the ratelimiting enzyme for polyamine synthesis, is increased in macrophages (Macs) in mouse and human Hp gastritis tissues, and that inhibition of ODC reduces gastritis and Hp colonization in mice. Our aim was to determine if ODC impairs host tolerance induced by Macs and promotes STAT1-mediated Th1/Th17 adaptive immune responses to Hp. Methods: Wild type (WT) and ODC+/C57BL/6 mice were infected with Hp SS1 for 4 months. We measured in gastric tissues: Hp colonization by qPCR, histological inflammation (0-12), TGFβ by Western blotting, and cytokines by Luminex assay. TGFβ in gastric Macs (GMacs) was measured by qPCR and immunofluorescencemicroscopy. Splenic Macs (SMacs) were isolated and activated with French-pressed Hp lysates (HPL); levels of TGFβ mRNA and nitric oxide (NO) production were measured by qPCR and Griess assay, respectively. Splenocytes, or co-culture of splenocytes depleted of Macs (SMacs-) with SMacs were activated with HPL for 48 h ± anti-TGF-β neutralizing antibody or the iNOS inhibitor 1400W, and IFNγ, IL17, IL-10, pSTAT1, and pSTAT3 were analyzed in CD4+ cells by flow cytometry. Results: In Hp-infected ODC+/versus WT mice, there was decreased histological gastritis (from 5.8 ± 0.3 to 3.2 ± 0.2; p , 0.05) and Hp colonization was reduced by 2 log orders ( p , 0.05). In SMacs from ODC+/-vs. WT mice, ex vivo activation with HPL increased TGFβ mRNA levels by 10-fold (p , 0.01) and NO production (from 6.2 ± 0.4 to 15.3 ± 0.9 μM; p , 0.01), but there was no difference in IL-10 expression. In HPL-stimulated splenocytes, IFNγ+ and IL-17+cells were increased in WT mice, but IL-10+ cells were increased in ODC+/mice. Pretreatment of splenocytes with anti-TGFβ antibody or 1400W before ex vivo activation increased IFN-γ+ and IL-17+, but not IL-10+ cells in ODC+/mice (Table 1). When WT SMacs were co-cultured with WT or ODC+/SMacs-, upon ex vivo stimulation there were increased IFN-γ+ cells, but when ODC+/SMacs were co-cultured with WT or ODC+/SMacs-, this did not occur. Pretreatment with anti-TGFβ or 1400W in the co-culture of ODC+/-SMacs with WT or ODC+/SMacsincreased IFN-γ+ cells (Table 2). In stomach tissue lysates from ODC+/vs. WT mice, IFN-γ and IL-17 levels were decreased, while IL-10 was increased. TGF-β mRNA and protein expression was increased in stomach tissues and in GMacs of ODC+/mice. In these studies, the pSTAT1/pSTAT3 ratio was decreased when IFN-γ and IL-17 were decreased and IL-10 was increased. Conclusions: During Hp infection, ODC inhibits TGF-β and NO production by Macs that impairs host immune tolerance manifested as a skewed adaptive (Th1/Th17) response, inappropriate inflammation, and persistent Hp colonization. Table 1

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 763: Hyperthermia promotes c-myc turnover and reduces ODC expression in colorectal cancer cells.

Abstract Hyperthermia therapy is an FDA-approved procedure that is used to improve the clinical response to radiation and chemotherapy in a variety of solid malignancies. This method involves raising the temperature of tumor-bearing tissues to 41-43°C, which is commonly achieved by using microwave or radiofrequency (RF)-emitting devices. Despite a multitude of treatment centers that are now providing this type of therapy, the cellular and molecular processes are not fully understood. Here, we report that that the oncogenic transcription factor, c-myc has a potentially important role in the anti-cancer mechanism of hyperthermia. By treating colorectal cancer cells (RKO cell line) with heat (incubation at 42°C), we observe a rapid decrease in c-myc levels that is not seen with other transcription factors. We have also observed a similar effect in other cell types, including lung (A549) and neuroblastoma (SH-SY5Y). It is already known that the turnover of c-myc protein is enhanced by phosphorylation at Thr58. Our Western blot data reveal that hyperthermia causes a significant increase in Thr58 phosphorylation, which is accompanied by a reduction in total c-myc levels. c-Myc is frequently deregulated in human cancers, where it promotes the expression of genes that drive tumor cell proliferation. To assess the role of c-myc target genes in the response to hyperthermia, we looked at one such target gene, ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. We see that the hyperthermia-induced degradation of c-myc is accompanied by a drastic reduction in ODC mRNA and protein levels. Using actinomycin D, we observe that heat has no effect on ODC mRNA stability, suggesting that the decrease in ODC is due to a reduction in gene transcription. In support of this, we find that silencing c-myc expression with siRNA inhibits ODC mRNA and protein levels by a similar amount as hyperthermia. We propose that the down-regulation of ODC caused by c-myc degradation may be an important part of the clinical response to hyperthermia, and future work will evaluate the effect of heat on cellular polyamine levels. Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Hyperthermia promotes c-myc turnover and reduces ODC expression in colorectal cancer cells. [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 763. doi:10.1158/1538-7445.AM2013-763

Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer

Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70–75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell.

Environmental pesticide exposure modulates cytokines, arginase and ornithine decarboxylase expression in human placenta

To evaluate the cytokine balance and enzymatic alterations induced by environmental pesticide exposure during pregnancy, this transversal study explored placentas derived from non-exposed women (control group-CG), and from women living in a rural area (rural group-RG), collected during intensive organophosphate (OP) pesticide spraying season (RG-SS) and during non-spraying season (RG-NSS). The exposure biomarkers blood cholinesterase and placental carboxylesterase (CaE) were significantly decreased in RG-SS. Among the cytokines studied IL-8, IL-6, TNFα, IL-10, TGFβ and IL-13, the expression frequency of IL-13 increased in RG-SS. Arginase and ornithine decarboxylase (ODC) enzymes were induced in syncytiotrophoblast and endothelial cells. Interestingly, the decrease in CaE activity was associated with arginase and ODC activity induction. These findings suggest that environmental pesticide exposure impacts the placenta by increasing the expression frequency of the anti-inflammatory cytokine IL-13, which may be related to the up-regulation of enzymes implicated in tissue repair.

Diabetes/high glucose induced arginase increases arterial smooth muscle cell proliferation and collagen synthesis/fibrosis through ornithine decarboxylase and ornithine aminotransferase pathways

Arginase can compete with nitric oxide (NO) synthase for their common substrate, L‐arginine, and its increased activity can decrease NO production. Arginase also provides ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT). We hypothesize that diabetes/high glucose (HG) induce vascular wall thickening and fibrosis through increased level of arginase‐derived ornithine available for ODC and OAT. We exposed rat aortic smooth muscle cells to HG (25 mM), which resulted in increased arginase activity (57%), cell proliferation (22%), and ODC mRNA (25%). Treatment with arginase inhibitor BEC (S‐(2‐boronoethyl)‐l‐cysteine) prevented these changes. We observed increased perivascular fibrosis (68%) in coronary arteries of 8‐week wild‐type (WT) diabetic mice compared to arginase I partial knockout (AI+/−) diabetic mice. Aorta from diabetic WT mice also exhibited impaired endothelium‐dependent relaxation (EDR) compared to control (maximum EDR: 32.9% vs 61.8%). EDR was largely maintained in aorta from diabetic AI+/− mice. Furthermore, adenoviral overexpression of arginase I in isolated aorta concomitantly increased ODC expression, implying an effect of arginase on ODC. In conclusion, arginase appears to have important roles in HG/diabetes‐induced vascular dysfunction, proliferation and fibrosis through ODC and OAT pathway.

Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.

Ornithine decarboxylase and glutamate decarboxylase 65 as prognostic markers of gallbladder malignancy: A clinicopathological study in benign and malignant lesions of the gallbladder

Ornithine decarboxylase (ODC) plays a critical role in cell proliferation and is overexpressed in a variety of cancers. Furthermore, γ-aminobutyric acid (GABA) content and glutamate decarboxylase (GAD) activity are increased in neoplastic tissues in colon and breast cancer. However, few studies have examined these molecules in gallbladder cancer specimens. We observed the expression levels of ODC and GAD65 in benign and malignant lesions of the gallbladder and investigated their clinicopathological significance for the first time. The expression levels of ODC and GAD65 in specimens from gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyps (n=15) and chronic cholecystitis (n=35) were detected using immunohistochemical methods. Kaplan-Meier survival and Cox regression analyses were carried out to explore the clinical and pathological correlations. The levels of positive staining of ODC and GAD65 were significantly higher in gallbladder adenocarcinoma than in peritumoral tissues, adenomatous polyps and chronic cholecystitis. The Kaplan‑Meier survival analysis and Cox regression analysis showed that the expression of ODC and GAD65 correlated significantly with the one-year survival rate and the mean survival time of the patients postoperatively. We conclude that the overexpression of ODC and GAD65 are significant in the carcinogenesis and progression of gallbladder adenocarcinoma. They may be important biological markers for the evaluation of biological behaviors and the prognosis of gallbladder adenocarcinoma.

Abstract CN04-03: Development of NSAID eflornithine combinations for treating cancer risk factors

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDS) have been found to be potent inhibitors of carcinogenesis in both preclinical models and in randomized controlled prospective clinical trials in humans. NSAIDS exert their anti-carcinogenic effects by inhibiting cyclooxygenases (COXs) involved in arachidonic acid metabolism and by COX-independent mechanisms. Empirical data indicates eflornithine (difluoromethylornithine or DFMO), an enzyme-activated inhibitor of ornithine decarboxylase (ODC) (Meyskens and Gerner, 1999), is one of the most potent agents known acting in combination with NSAIDS to inhibit carcinogenesis in rodent models (Steele and Lubet, 2010). At least part of the rationale for combining NSAIDS with eflornithine for inhibition of carcinogenesis is that eflornithine inhibits the activity of ODC, the first enzyme in polyamine synthesis, while NSAIDS activate the spermidine/spermine acetyltransferase (SAT1), which targets polyamines for export by specific solute carrier transporters (Gerner and Meyskens, 2009). Thus, NSAIDS and eflornithine both reduce tissue levels of the growth-associated polyamines, but by complementary mechanisms. A clinical trial of the combination of eflornithine and the NSAID sulindac showed dramatic treatment-associated reductions of metachronous colorectal adenomas in patients with prior sporadic colorectal polyps (Meyskens et al., 2008). Several clinical trials in progress or soon to commence will further test the hypothesis that NSAID eflornithine combinations can successfully treat cancer risk factors in patients with specific cancers, or risk of cancer. One group of clinical trials involves patients with neuroblastoma (NB). Patients with poor prognosis NB often have tumors in which MYCN is overexpressed. Preclinical data indicates that MYCN as well as c-MYC drive expression of ODC and other genes in the polyamine pathway, and that inhibiting this pathway with eflornithine suppressed carcinogenesis in mouse models of NB (Hogarty et al., 2008). Likewise, COX-2 is expressed in NB tumors and cell lines, and COX-2 inhibitors such as celecoxib can suppress the growth of NB xenografts (Ponthan et al., 2007). The Neuroblastoma and Medulloblastoma Translational Research Consortium (NMTRC) and the New Approaches to Neuroblastoma Therapy (NANT) group are conducting clinical trials to evaluate the safety and efficacy of eflornithine alone or in combination with NSAIDS and other agents in patients with high risk NB. The NMTRC is conducting an especially novel prevention trial of eflornithine in patients with high risk NB in remission (NCT01586260). Eflornithine NSAID combinations are also being evaluated in other MYC-associated diseases. Familial adenomatous polyposis (FAP) is a genetic syndrome associated with increased risk of colon cancer and other neoplasia and is caused by mutation/deletions in the adenomatous polyposis coli (APC) tumor suppressor gene. MYC mediates intestinal tumorigenesis (Ignatenko et al., 2006) and combinations of eflornithine and NSAIDS are potent inhibitors of intestinal carcinogenesis (Ignatenko et al., 2008) in murine models of FAP. Notable is the change in clinical management of FAP patients over the past two decades. FAP is now managed primarily by surgery, with duodenal polyposis and desmoid disease constituting two current significant clinical problems. An international consortium will be evaluating the combination of eflornithine and sulindac in adult patients with FAP, using time to FAP-related events as the primary outcome (NCT01483144). This same combination will be evaluated in patients with prior sporadic colon cancer in a study to be conducted by a national cooperative group (S0820, Adenoma and second primary prevention trial, NCT01349881) (Rial et al., 2012). These and other trials have been designed to include assessment of a range of biological correlates, including genetic (Zell et al., 2010), tissue (Thompson et al., 2010) and urinary markers (Hiramatsu et al., 2005) of disease prognosis and prediction of treatment responses, including therapy-associated toxicities. Citation Format: Eugene W. Gerner. Development of NSAID eflornithine combinations for treating cancer risk factors. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN04-03.

P067 JAK2 V617F mutation causes cytokine-independent growth and tumorigenesis – Roles of STAT5 and c-Myc

Introduction JAK2 is an essential signal transducer for various cytokine signaling including IL-3 or Epo. Mutation at V617F of JAK2 promotes cell growth and prevents apoptosis which causes a variety of myeloproliferative neoplasms (MPNs). How the JAK2 V617F mutation causes dysregulated proliferation, resistance to anti-tumor drugs and tumorigenesis in nude mice is to be investigated. Methods Ba/F3 cells harboring JAK2 V617F were established to study the constitutive activation of JAK2 and cytokine-independent growth. Tumorigenesis was tested by the inoculation of JAK2 V617F mutant in nude mice. DNA array was done to search genes expressed predominantly by JAK2 V617F and STAT5. Results (1) JAK2 V617F mutant caused aberrant activation of STAT5 and Akt when EpoR was expressed, which is critical for the JAK2 V617F-induced anti-apoptotic and oncogenic activities. (2) In search of genes induced by JAK2 V617F mutant, we observed significant induction of c-Myc. (3) While the phosphorylation at Thr58 by GSK-3b induced the proteasome-mediated degradation of c-Myc, GSK-3b was inactivated in JAK2 V617F mutant, which resulted in the enhancement of c-Myc expression. The enforced expression of c-Myc accelerated not only the cell proliferation but also apoptotic cell death; however, the inhibition of GSK-3b completely inhibited c-Myc-induced apoptosis. (4) JAK2 V617F mutant also induced expression of a target gene of c-Myc, ornithine decarboxylase (ODC) known as a rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO) prevented the proliferation of the JAK2 V617F mutant cells. Furthermore, administration of DFMO effectively delayed tumor formation in nude mice, resulting in a prolonged survival. Conclusion JAK2 V617F mutant caused aberrant activation of Akt and STAT5 pathways. ODC expression induced by c-Myc is critical for JAK2 V617F mutant-driven transformation and that targeted disruption of c-Myc-ODC axis may provide therapeutic utility for the treatment of MPNs. STAT5 and c-Myc have critical roles for JAK2 V617F mutant-induced tumorigenesis, which help clarify the mechanism how JAK2 V617F mutant causes MPNs.

Abstract 1989: The effect of resveratrol combined with DFMO or DenSpm on MCF-7 breast cancer cells and MCF-10a non-cancerous cells

Abstract Resveratrol, (3,5,49-trihydroxy-trans-stilbene), was first reported to act as a chemo-preventative anticancer compound in mice by Jang et al. in 1997. Resveratrol has been shown to induce apoptosis and reduce both cell viability and mitotic index in many cancer cell lines, including MCF-7. The current work extends these studies to determine its effects on glucose utilization and enzymes involved in polyamine metabolism. Glucose uptake was found to be reduced in MCF-7 cells treated with resveratrol. Work in other labs has shown resveratrol to sensitize cancer cells to other chemotherapeutic compounds. In the present study, the effects of combinations of resveratrol with compounds that affect polyamine metabolism, DFMO (difluoromethylornithine) and DenSpm (N1, N11-diethylnorspermine), were tested. Both DFMO and DenSpm were found to reduce cell proliferation and mitotic index in MCF-7 cell lines and more importantly, a synergistic effect was observed when these compounds were used in combination with resveratrol. In this investigation resveratrol was also found to induce apoptosis and reduce cell viability, mitotic index, and glucose uptake in non-cancerous, transformed mammary epithelial cells (MCF-10a). Similar findings have been reported with other non-cancerous cells including intestinal smooth muscle cells (Garcia et al., 2011). The combination of resveratrol and DFMO also reduced glucose uptake, proliferation, and mitotic index in this non-cancerous cell line (MCF-10a). These compounds were found to affect the expression of ODC (ornithine decarboxylase) and SSAT (spermidine/spermine acetyl transferase), enzymes involved in polyamine metabolism, and other proteins involved in cell cycle regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1989. doi:1538-7445.AM2012-1989

Abstract 2173: Heterozygosity for Mtap accelerates lymphomagenesis in Emu-Myc and Pten +/− mice

Abstract The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. Loss of MTAP is associated with elevated expression of ornithine decarboxylase (ODC), a key enzyme in the synthesis of polyamines and a known oncogene. Here, we have examined if heterozygosity for a null mutation in Mtap could accelerate tumor development in two different mouse cancer models, Eα-myc transgenic and Pten+/−. We found that the median age of tumor formation in both models was significantly accelerated in Mtap+/− compared to Mtap+/+ mice. In Eα-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage and these tumors tended to be of higher grade and have higher expression levels of ODC protein compared to those observed in control Emu-myc Mtap+/+ mice. Supprisingly, examination of Mtap mRNA and protein in highly lymphoma infiltrated tissue in heterozygous animals indicates that >80% of the tumors maintain expression of the Mtap allele. Our findings show that germline inactivation of a single Mtap allele enhances lymphomagenesis in both Emu-myc and Pten+/− mice and is associated with elevated levels of ODC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2173. doi:1538-7445.AM2012-2173