bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5366: Chronic, low-dose treatment with sulforaphane inhibits growth of breast cells.
Authors: Silvia Ashley L. , Browne Eva P. , Arcaro Kathleen F.
Published: August 2014

Epidemiological data indicate that dietary consumption of cruciferous vegetables may reduce the risk of many cancers including breast cancer. Cruciferous vegetables contain high levels of glucosinolates; stable phytochemicals that when metabolized produce cancer-preventing active isothiocyanates. Isothiocyanates, such as sulforaphane (SFN) found in broccoli sprouts, are thought to prevent tumorigenesis through epigenetic mechanisms controlling the expression of tumor-related genes. Ingestion of broccoli sprouts results in rapid distribution of SFN to breast epithelium, however, the effect of chronic exposure to low levels of SFN on the growth of breast cells has not been fully explored.

In the present study we assessed the effect of SFN on the growth of an immortalized breast cell line (MCF10A), and two breast cancer cell lines; the estrogen receptor positive MCF-7 cell line, and the estrogen receptor negative TMX2-28 cell line. We first determined the seeding densities for each cell line that resulted in 80% confluence after three days of growth in T-25 flasks: MCF10A (1 x 106), MCF-7 (8 x 105), and TMX2-28 (5 x 105) per 5 mL. Next, cells from each line were plated at the predetermined seeding densities in T-25 flasks and treated with either SFN (5 μM) or vehicle (0.02% DMSO) for up to 40 days. Every third day, cell cultures were photographed, after which the cells were resuspended in culture medium, counted using a hemocytometer, and the predetermined numbers of cells (seeding numbers) were returned to T-25 flasks and treated with SFN or vehicle. Cells not needed for seeding the flasks were archived for future analysis of gene expression and DNA methylation.

Treatment with SFN decreased cell growth. After 22 days of treatment with SFN, mean proliferation of both the MCF10A and MCF-7 cell cultures was inhibited by 62% (t = 2.8; p = 0.02) and 72% (t = 2.6; p = 0.02), respectively. In contrast, the decrease in proliferation of TMX2-28 cells treated with SFN was not statistically significant (t = 1.2; p = 0.255) from the vehicle control at 22 days. However, by day 40, TMX2-28 cell cultures treated with SFN showed a significant decrease in mean proliferation 36% (t = 2.2; p = 0.04). Ongoing studies are examining the epigenetic mechanisms by which SFN decreased cell proliferation.

Citation Format: Ashley L. Silvia, Eva P. Browne, Kathleen F. Arcaro. Chronic, low-dose treatment with sulforaphane inhibits growth of breast 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 5366. doi:10.1158/1538-7445.AM2013-5366
Abstract 5376: Expression of histone deacetylases (HDACs) 1, 2, 3 and 7 in pancreatic cancer cell lines: The role of indole-3-carbinol.
Authors: Lyn-Cook Beverly D. , Pang Li , Lyn-Cook Lascelles E. , Word Beverly , Hammons George
Published: August 2014

Pancreatic cancer has the poorest prognosis of most cancers, mainly due to its resistant phenotype. Early or late biomarkers are greatly needed in this cancer. Scientists have shown that epigenetic mechanisms may be involved in the etiology and progression of pancreatic cancer. Epigenetic changes involve modification of the DNA through hyper- and hypo-methylation, histone modification and modulation in expression of microRNAs. Histone deacetylases modify tails on the histones, which can result in repressive chromatin formation and inactivation of specific genes. This study, using real-time PCR, examined the level of HDACs 1, 2, 3 and 7 in four pancreatic cancer cell lines (BXPC, CFPAC,ASPC and MIA). The highest level of expression of HDAC 1 and 3 was found in CFPAC and MIA cells compared to BXPC and ASPC cells. Indole-3 carbinol, in previously published studies from our laboratory, have shown several anti-cancer activities, including the re-expression of p16 in MIA cells (Lyn-Cook et al, 2010 Anticancer Res., 30:4907-4914). The current study demonstrated that indole-3-carbinol at concentrations of 20 μM and 100 μM significantly decreased expression of HDAC 3 by 2-fold and 3-fold in MIA cells, respectively. Results further showed that indole-3 carbinol decreased expression of HDAC 1 in CFPAC cells and HDAC 7 in BXPC cells by 2-fold. No change in expression was shown in any of the cell lines on HDAC 2. This study showed that indole-3-carbinol may exert histone deacetylase inhibitory effects. Further studies are needed to confirm these results. Specific HDACs as potential targets for pancreatic cancer are growing among the scientific community. HDACs are known to be regulators of growth, differentiation and cell death and dysfunction of transcriptional repression mediated by HDACs may lead to the development of cancer.

Citation Format: Beverly D. Lyn-Cook, Li Pang, Lascelles E. Lyn-Cook, Beverly Word, George Hammons. Expression of histone deacetylases (HDACs) 1, 2, 3 and 7 in pancreatic cancer cell lines: The role of indole-3-carbinol. [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 5376. doi:10.1158/1538-7445.AM2013-5376
Abstract 5377: Profiling of aurora kinase signaling in microdissected gastric cancer samples indicates a significant increase in histone deacetylase 4 (HDAC4).
Authors: Colarossi Lorenzo , Memeo Lorenzo , Liotta Lance A. , Espina Virginia , Mueller Claudius
Published: August 2014

Introduction & Methods: Gastric cancer represents one of the leading causes of cancer related death in the world second only to lung cancer resulting in 800,000 deaths per year. Aurora kinases, a family of serine/threonine kinases essential for cytokinesis and chromosome segregation, are frequently over-expressed in various cancer types. Moreover, Aurora kinase has recently been shown to directly regulate epigenetic changes (Guise et al., Mol Cell Proteomics. 2012 Nov;11(11):1220-9). Aurora kinase signaling may also impact E-cadherin function via a GSK3-α/β induced increase in β-catenin. E-cadherin deregulation has been implicated in gastric cancer, especially in diffuse types where it is mutated in many cases.

We have profiled Aurora kinase signaling pathway activation and its effects on epigenetic and cytoskeletal changes in gastric cancer. Human gastric cancer tumor samples (n=10; 4 diffuse, 5 intestinal, 1 undifferentiated adenocardinoma) and patient matched normal mucosa were procured by laser capture microdissection. Reverse phase protein microarrays were used to quantify total and post-translationally modified proteins in cell signaling pathways directly and indirectly related to Aurora kinase.

Results: We found a significant increase in histone deacetylase 4 levels (HDAC4, p<0.0001) concomitant with increased phosphorylation of Aurora kinase (p<0.1) and a decrease in phosphorylated GSK3-α/β (p<0.05) in gastric tumor epithelia compared to patient matched normal mucosa. Gastric cancer samples of diffuse type showed a significantly reduced expression of E-Cadherin (p<0.05) as compared to intestinal type gastric cancer. In contrast, patient samples with lymph node metastasis (n=6) were associated with an increased level of phospho-GSK3-α/β in both normal mucosa (p<0.05) and matched tumor epithelia (p<0.1).

Conclusions: To our knowledge, this is the first time that increased levels of HDAC4 have been reported in gastric cancer. The demonstrated increase in Aurora kinase phosphorylation in gastric tumor epithelia may provide a mechanistic link to the observed HDAC4 upregulation.

Citation Format: Lorenzo Colarossi, Lorenzo Memeo, Lance A. Liotta, Virginia Espina, Claudius Mueller. Profiling of aurora kinase signaling in microdissected gastric cancer samples indicates a significant increase in histone deacetylase 4 (HDAC4). [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 5377. doi:10.1158/1538-7445.AM2013-5377
Abstract 5384: Hrev107 enhances prostaglandin D2 synthase-mediated cancer cells suppression in the testis.
Authors: Tsai Fu-Ming , Wu Chang-Chieh , Shyu Rong-Yaun , Jiang Shun-Yuan
Published: August 2014

Hrev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family which includes HREV107, Retinoid-inducible gene 1 (RIG1), and HRASLS. The protein in this family contains an NC domain, with unknown function at the N-terminus, and a hydrophobic membrane-anchoring domain at the C-terminus. HREV107 has shown that expressed at high levels in differentiated tissues of post-meiotic testicular germ cells but not in human testicular germ cell tumors. Prostaglandin D2 (PGD2) has shown that can induce SOX-9 nuclear translocation and subsequent Sertoli cell differentiation. In addition, expression of the Prostaglandin D2 synthase (PTGDS) has been detected in the cellular lineage. This gives rise to the Sertoli cells, and the Sertoli cells appear to express the PTGDS only during the VI-VIII stages of the spermatogenic cycle after spermiation. These results suggested that PGD2 may play an important role in the testis differentiation. Our previous result showed that RIG1 (as RIG1 is expressed in the human species only) can interact with PTGDS. We further examined the effect of mouse Hrev107 on the growth of mouse testicular cells. When Hrev107 was expressed in TM3 or TM4 mouse testicular cancer cells, the cell migration and invasion were inhibited. Further, we found PTGDS was co-localized with Hrev107 in TM4 testis cells. In Hrev107-expressing NT2/D1 cells, elevated levels of PGD2, cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This result indicated that Hrev107 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased h-REV107-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated Hrev107-mediated suppression of migration and invasion. These results suggest that Hrev107 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, Hrev107 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion.

Citation Format: Fu-Ming Tsai, Chang-Chieh Wu, Rong-Yaun Shyu, Shun-Yuan Jiang. Hrev107 enhances prostaglandin D2 synthase-mediated cancer cells suppression in the testis. [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 5384. doi:10.1158/1538-7445.AM2013-5384
Abstract 5390: Chemical disruption of Wnt protein fatty acylation as an anti-cancer therapeutic strategy.
Authors: Tuladhar Rubina , Lum Lawrence
Published: August 2014

The secreted Wnt signaling molecules are associated with a broad range of cancers and represent high priority therapeutic targets. Recently identified small molecules that disrupt Wnt biosynthesis by disabling the extracellular acyltransferase Porcupine (Porcn) form the basis of a novel targeted anti-cancer therapeutic strategy. Porcn is a founding member of a sixteen gene family known as Membrane-Bound O-Acyltransferases (MBOATs) that likely control the production of a broad range of disease-relevant signaling molecules. A deeper understanding of the chemical-protein interaction that confers Porcn inhibitors with selectivity would facilitate the engineering of improved Porcn inhibitors and molecules with activity against other MBOAT family members. Using synthetic fatty acyl analogues that allow direct labeling of Wnt proteins and chemical inhibitors of fatty acid metabolic enzymes, we have uncovered that Porcn utilizes a monounsaturated 16-carbon fatty acid as a substrate. Chemical disruption of stearoyl-CoA desturase (SCD) that converts palmitate to palmitoleate blocks Wnt protein production and activity thus revealing another potential chemical strategy for inhibiting Wnt-dependent cancerous growth. The inability of several other Porcn family members with known protein substrates to utilize palmitoleate may account for the selectivity of Porcn classes of inhibitors. Our study expands the number of drug targets to include those enzymes that control the production of secreted signaling molecules and reveals a direct influence of cellular metabolic status on coordinated and cancerous cell growth regulation.

Citation Format: Rubina Tuladhar, Lawrence Lum. Chemical disruption of Wnt protein fatty acylation as an anti-cancer therapeutic strategy. [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 5390. doi:10.1158/1538-7445.AM2013-5390
Abstract 5392: Metabolic profiling of castrate-resistant prostate cancer reveals novel role for bile acids in driving castration resistance.
Authors: Chakravarthy Divya , Rivas Paul , Keppler Brian , Ruan Jianhua , Ghosh Rita , Kumar Addanki Pratap
Published: August 2014

The 5-year survival for metastatic castrate-resistant prostate cancer patients is less than 30% despite significant progress in the understanding of prostate cancer biology and development of novel therapeutic agents. A major contributing factor for the observed low survival rate of patients with castrate resistant disease is the lack of knowledge regarding metabolic alterations and their underlying contributions during development of castrate-resistant phenotype. Studies conducted in our laboratory and others identified a potential role for 2-methoxyestradiol (2-ME2) to prevent prostate cancer development and progression through inhibition of the anti-apoptotic protein FLIP. The goal of the current study was to identify biochemical changes in response to castration and treatment with 2-ME2 in serum from transgenic adenocarcinoma of mouse prostate (TRAMP) mice using mass spectrometry based global profiling. We identified a total of 54 biochemicals of which 16 increased and 38 decreased in castrated animals compared to sham-castration. Treatment of sham-castrated animals with low and high doses of 2-ME2 altered 91 and 145 biochemicals respectively. On the other hand treatment of castrated animals modulated 89 and 106 biochemicals. Cumulative analysis of these data also identified alteration of 60 biochemicals associated with castration effect, 149 with treatment and 70 interactions between castration and treatment effects. Castration affected metabolites involved in variety of metabolic pathways including lipid, oxidative stress, energetics and bile acid. Given the data showing enhanced expression of FLIP in castrate-resistant prostate tumors and upregulation of bile acids in patients undergoing androgen deprivation therapy, we examined the activation of FLIP in prostate cancer cells in response to deoxycholic acid (DCA). Our data suggests that transcriptional activity of FLIP was higher in PC-3 cells treated with DCA. To the best of our knowledge, this is the first report demonstrating global metabolomic profiling of serum in response to castration and provide a framework for therapeutic targeting of bile acid metabolism. Supported by NIH CA 135451 (APK).

Citation Format: Divya Chakravarthy, Paul Rivas, Brian Keppler, Jianhua Ruan, Rita Ghosh, Addanki Pratap Kumar. Metabolic profiling of castrate-resistant prostate cancer reveals novel role for bile acids in driving castration resistance. [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 5392. doi:10.1158/1538-7445.AM2013-5392

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5400: Exercise activates AMP-activated protein kinase in breast cancer cells.
Authors: Højfeldt Grith W. , Dethlefsen Christine , Pedersen Bente K. , Hojman Pernille
Published: August 2014

Objective: Cancer cells are characterized by a changed cellular metabolism, favoring glycolysis. AMPK, a serine/threonine kinase regulates cellular metabolism, and induces fatty acid oxidation and has in regard to this, been shown to suppress tumor cell growth. During physical activity, which is associated with a reduced cancer risk, working muscles secrete myokines, known to activate AMPK. This could indicate a connection between AMPK activity, myokine release from the working muscle, and an anti-cancer response. We hypothesize that exercise-induced myokine secretion causes an anti-cancer response via AMPK activation. Methods: Exercise-induced AMPK activation was studied in two models, 1) A cell study using the two breast cancer cell lines, MDA-MB-231 and MCF-7, incubated with exercise-conditioned serum, representing a myokine pool. Serum was collected from 9 females during a 2 hours cycling bout, and during a subsequent 3 hours rest. Following incubation, cell viability and AMPK phosphorylation was determined in the cells. 2) An In Vivo model, where the two cell lines were injected into nude mice. When the tumors reached 6 mm in diameter, the mice were subjected to 1 hour forced swimming bout, after which the tumors were dissected and analyzed for pAMPK content. Results: 1) Cell study: Two hours of cycling induced a 6-fold increase in serum IL-6, as expected. Incubating cancer cells with this post-exercise serum resulted in a significant reduction in the viability of MCF-7 cells, while no effect on the viability of MDA-MB-231 cells were observed. Preliminary data show increased AMPK phosphorylation in both MDA-MB-231 and MCF-7 cells after incubation with 5% exercise-conditioned serum. 2) In vivo study: 1 hour of swimming resulted in significantly increased AMPK phosphorylation in the MCF-7 tumors. Conclusion: Our preliminary data indicate that exercise-induced factors can cause decreased viability and increased AMPK activity in MCF-7 cells, while MDA-MB-231 cells need further investigation. Acknowledgement: The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National research Foundation (#02-512-55). This study was further supported by the Danish Medical Research Council and the Lundbeck Foundation.

Citation Format: Grith W. Højfeldt, Christine Dethlefsen, Bente K. Pedersen, Pernille Hojman. Exercise activates AMP-activated protein kinase in breast 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 5400. doi:10.1158/1538-7445.AM2013-5400
Abstract 5402: Synergism of Metformin and DFMO on colon cancer proliferation and migration.
Authors: Zhang Yanping , Rodriguez Luz M. , Lubet Ronald , Hsueh Eddy C.
Published: August 2014

Introduction: Metformin has been shown to inhibit cancer cell growth via activation of MAP kinase with resultant inhibition of mTOR signaling pathway. ODC, a key enzyme in polyamine biosynthesis, was found to be overexpressed in colon cancer. We determined the effect of metformin in combination with DMFO, an ODC inhibitor, on tumor proliferation and migration in human colon cancer cells. Methods: Human colon cancer cells, HCT 116 and HT 29, were treated with Metformin, DFMO, or combination. Cell proliferation assay was performed using Cell Titer Blue proliferation assay. Cell migration assay was performed by seeding colon cancer cells in 24-well plate. After overnight incubation, an artificial homogenous wound was created onto the monolayer with a plastic micropipette tip. After wounding, the migration of cells into the wound was observed up to 72 hours at various time points. Quantitative RT-PCR for ODC1 detection was performed using Assay-on-Demand Hs00159739-ml. GAPDH served as control. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1. Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant. Results: There was dose-dependent (1-10 mM for DFMO and 1-20 mM for metformin) time-dependent (up to 96 hours) inhibition of cell proliferation and migration with either metformin or DFMO in both HCT 116 and HT 29 cells. Synergistic inhibition was observed with the combination treatment (5 mM DFMO and 5 mM Metformin) on both cell migration and proliferation at 24 hours compared with either agent alone. There was synergistic inhibition of ODC expression with the combined treatment compared with DFMO alone at 24 hours (p<0.01). The combination treatment also lead to significant enhancement of AMPK activation and increased phosphorylation of AMPKα at Thr-172 compared with metformin alone (p<0.01). The combination treatment resulted in significant inhibition of mTOR signaling with decreased phosphorylation of mTOR, p70S6K and 4E-BP1 in treated cancer cells, when compared with individual drug treatments. Inhibition of proliferation and migration was significantly greater in the mismatch-repair defective HCT 116 cells than HT 29. Conclusion: We have observed a synergistic effect of metformin and DFMO in suppressing human colon cancer proliferation and migration. The synergism reflects enhanced inhibition of both polyamine synthesis and mTOR signaling pathways by the drug combination.

Citation Format: Yanping Zhang, Luz M. Rodriguez, Ronald Lubet, Eddy C. Hsueh. Synergism of Metformin and DFMO on colon cancer proliferation and migration. [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 5402. doi:10.1158/1538-7445.AM2013-5402
Abstract 5405: Limiting glucose availability with metformin increased reactive oxygen species, decreased cell survival, and promoted apoptosis in human and mouse pancreatic cancer cells.
Authors: Yan Guang , Freeman Theresa , Lanza-Jacoby Susan
Published: August 2014

Background: Recently, we found that calorie restriction (CR) protected K-ras G12D; Pdx-1Cre mice against the progression of advanced PanIn lesions and pancreatic cancer. Since CR limits glucose availability, the CR mimetic metformin is also potential approach to prevent pancreatic cancer. Recent studies found that metformin reduced the incidence of pancreatic cancer in diabetic patients. However, it is not clear how metformin promotes the anticancer effects. One potential mechanism is by altering reactive oxygen species (ROS) and autophagy. In this in vitro study, we explore the role of ROS and autophagy in mediating the growth-inhibitory effects of metformin by treating pancreatic cancer cells and normal pancreatic ductal cells with metformin.

Methods: We acclimated MiaPaca and KP mouse pancreatic tumor cells from K-ras G12D; Pdx-1Cre mice to physiological concentrations of glucose and then treated cells cells with metformin with and without the antioxidant N-acetyl cysteine (NAC) or chloroquine (autophagy inhibitor) for 24h. Cell viability, ROS production, and apoptosis were measured.

Results: Metformin decreased viability of MiaPaca and KP cells in a concentration-dependent manner in comparison to untreated cells; metformin did not have any effect in HPNE human pancreatic normal epithelial cells. Pretreating MiaPaca and KP cells with NAC increase survival and decreased apoptosis, which suggests that ROS promotes the growth-inhibitory and apoptotic effect of metformin. Metformin increased ROS formation in MiaPaca and KP cells but not in HPNE cells. Cell survival increased and apoptosis decreased when MiaPaca cells were pretreated with chloroquine. Our findings suggest that ROS and autophagy may mediate the cytotoxic effects of metformin in MiaPaca and KP pancreatic cancer cells.

Conclusion: In summary, metformin may be a potential chemoprevention agent against pancreatic cancer without cytotoxic effects in normal pancreatic ductal cells.

Citation Format: Guang Yan, Theresa Freeman, Susan Lanza-Jacoby. Limiting glucose availability with metformin increased reactive oxygen species, decreased cell survival, and promoted apoptosis in human and mouse pancreatic 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 5405. doi:10.1158/1538-7445.AM2013-5405
Abstract 5416: Metabolic control of the prometastatic protein CD147.
Authors: Papathanassiu Adonia E. , Vu Hong A.
Published: August 2014

Apart from a recent report suggesting that hypoxia upregulates CD147, little is known about the mechanisms responsible of the tumoral overexpression of CD147, a transmembrane and secretory glycoprotein known to promote metastasis through multiple mechanisms. Here, we report that the total and surface concentration of CD147 appear to be under metabolic control. Glucose (Glc) deprivation in MDA-MB-231 and MDA-MB-435 tumor cells leads to a decrease in the levels of total CD147 and a dramatic increase in the expression of cell surface CD147. A similar mobilization of CD147 to the plasma membrane was observed with glutamine (Gln) deprivation in MDA-MB-435 cells; when the cells were deprived of both nutrients (Glc and Gln) a dramatic increase (∼6-fold) in the cell surface expression of CD147 was observed. Plasma membrane associated CD147 is known to promote metastasis through homotypic and heterotypic interactions between cancer and stromal cells. An 80% reduction in the membrane found CD147 was observed when Gln-deprived cells were treated with ERG-240, a small molecule that inhibits the cytosolic form of BCAT, the enzyme responsible for the reversible transamination of leucine (Leu). On the other hand, Leu deprivation was shown to decrease CD147 by 6-7 fold in both cancer cell lines tested here. Treatment of the cells with ERG-240 partially reversed the increase in the levels of CD147 seen after supplementation of the Leu-deprived cells with 105 mg/L Leu. CD147 is known to possess 3 N-glycosylation sites and to exist in forms with various degrees of glycosylation; the fully glycosylated protein is associated with an increased prometastatic potential. Inhibitors of N-glycosylation, such as 2-deoxyglucose (2DG), are known to possess anticancer properties. In the present study, treatment of MDA-MB-231 cells in vitro with 10 mM 2DG led to 80% reduction of the cell surface expression of highly glycosylated CD147 without the concomitant reduction in the total concentration of CD147. In contrast, treatment of athymic female nude mice, bearing orthotopically implanted MDA-MB-231 tumors, with 125 mg/Kg 2DG twice a week led to tumors that expressed markedly increased levels of the highly glycosylated CD147 variant (Control/Treated or T/C=2.3, p=0.01). The increased expression of the protein in the 2DG-treated tumors was accompanied with an increase in the levels of circulating human CD147 (T/C=1.7, p=0.01) normalized per tumor volume. Collectively, the data point towards a metabolic control of CD147 and suggest that inhibition of tumor growth due to nutrient deprivation may initiate pathways that promote metastasis.

Citation Format: Adonia E. Papathanassiu, Hong A. Vu. Metabolic control of the prometastatic protein CD147. [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 5416. doi:10.1158/1538-7445.AM2013-5416
Abstract 5417: HIF1α is a master regulator with the facilitate of demethylation for the hexokinase 2 expression during cancer progression.
Authors: Kim Hyemi , Lee Hyun G. , Lee Jae Myun , Park Jeon Han
Published: August 2014

Hexokinase 2 (HK2) is a key enzyme of aerobic glycolysis, known as the Warburg effect. A recent report showed that the HK2 expression in glioblastoma multiforme correlates with the progression of cancer and the survival of patients. The regulation of HK2 expression in cancer cells has been explored for many decades, however the precise mechanism is not yet identified. HK2 gene has a 4-kb promoter, which contains the potential responsive elements for glucose, insulin, glucagon, TPA, cAMP and HIF1 α. However, methylation of the promoter region makes normal cells nearly silent in HK2 expression. A study showed the potential contribution of demethylation of promoter for the HK2 expression during cancer progression.

In this study, we tried to identify the master regulator of HK2 expression. We performed methylation beadchip array for the comparison of methylation status of HK2 gene between normal tissues and hepatocellular carcinoma (HCC). The 5’-UTR and 3’-UTR are densely methylated in normal tissues, but significantly demethylated in HCC. Moreover, we identified new hypoxia responsive element (HRE) through the deletion and site-directed mutagenesis study with 2-kb promoter of human HK2. The mutation of new HRE diminished the luciferase activity. Specific interaction between HIF1 α and HRE were identified by ChIP and EMSA. Then, we showed that demethylation by 5-Aza-deoxycytidine could contribute to the HK2 expression in hypoxic condition.

In conclusion, we revealed the mechanism of HK2 expression and hypoxia is one of the major regulator among pre-known regulators. The sequential events, demethylation and exposure to hypoxic environments are the main requisite for the HK2 expression during the cancer progression.

Citation Format: Hyemi Kim, Hyun G. Lee, Jae Myun Lee, Jeon Han Park. HIF1α is a master regulator with the facilitate of demethylation for the hexokinase 2 expression during cancer progression. [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 5417. doi:10.1158/1538-7445.AM2013-5417
Abstract 5419: Thiamine deficiency induces HIF-1 α stabilization and target gene expression.
Authors: Sweet Rebecca , Lim Lauren , Zastre Jason
Published: August 2014

Introduction: Thiamine deficiencies have been reported in a number of common pathologies including diabetes, neurodegenerative disorders and in particular, cancer. Deficiencies can arise due to drug mediated reductions in cellular availability of thiamine or due to limited diffusion into solid tumors. Biochemically, a metabolic consequence of thiamine deficiency is a reduction in enzyme activity and expression of pyruvate dehydrogenase complex (PDH), which converts pyruvate to acetyl-CoA. Evidence suggests that trans-activation of hypoxia inducible factor 1-alpha (HIF-1α) regulated genes is mediated through stoichiometric changes in the metabolic intermediate pyruvate, independent of low oxygen termed pseudo-hypoxia. The resulting dysfunction of PDH and accumulation of pyruvate during TD may facilitate a pseudo-hypoxic state and mediate trans-activation of HIF-1α regulated genes. Therefore, we hypothesize that thiamine deficiency induces a pseudo-hypoxic state resulting in a stabilization of HIF-1α.

Methods: The impact of TD treatments on cellular pyruvate levels was determined after 48h of TD treatment using a pyruvate assay. Protein expression of HIF-1α was assessed in nuclear lysates of SK-N-BE cells treated with 1% O2 and TD for 12, 24, and 48h by Western blot. Expression of the known HIF-1α regulated gene, GLUT1 was measured by qRTPCR after 12, 24 and 48h hypoxia (1% O2) and thiamine depletion.

Results: An approximately 2 fold increase in cellular pyruvate was observed after 48h of TD exposure. A corresponding increase in HIF-1α protein expression in both 1% O2 and TD was found after 48h exposure. SK-N-BE cells demonstrated a 6 and 10 fold increase in expression of GLUT1 and SLC19A3, respectively after 48h of 1% O2 exposure. TD treatment exhibited an approximately 2 fold increase in expression of both GLUT1 and SLC19A3 after 48h treatments.

Conclusions: An increase in pyruvate production and the induction of HIF-1α protein expression after TD treatment suggests the role of TD acting as a feed forward mechanism for HIF-1α stabilization and gene regulation. These results demonstrate nutritional regulation of the pro-oncogenic transcription factor, HIF-1α and an important connection of cellular thiamine to adaptive glycolytic metabolism.

Citation Format: Rebecca Sweet, Lauren Lim, Jason Zastre. Thiamine deficiency induces HIF-1 α stabilization and target gene expression. [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 5419. doi:10.1158/1538-7445.AM2013-5419
Abstract 5420: Normal mammary epithelial mitochondria introduced into human breast cancer cells results in inhibition of proliferation and increased drug sensitivity of the MCF-7 breast cancer cell line.
Authors: Elliott Robert L. , Jian Xian P. , Head Jonathan F.
Published: August 2014

Warburg in the 1920s postulated that mitochondrial dysfunction alters metabolism in cancer. He observed in cancer cells aerobic glycolysis, which is the conversion of glucose to lactic acid in the presence of oxygen, the so called “Warburg effect” [1]. Mitochondrial dysfunction of cancer cells includes increased aerobic glycolysis, elevated levels of ROS, decreased apoptosis, and resistance to chemotherapeutic agents. We reported on the ultrastructural observations of mitochondria in human breast carcinoma cells [2]. There were three distinct groups: (1) mitochondria absent, (2) mitochondria present, (3) mitochondria present but sparse and abnormal. Tumors in the absent group were more anaplastic, aggressive, and resistant to treatment. We hypothesized that the introduction of normal mitochondria into cancer cells might restore mitochondrial function and inhibit cancer cell growth, and reverse chemoresistance. First we tested if mitochondria of normal mammary epithelial MCF-12A cells could enter into cultured human cancer cells. Then we determined if introducing normal mitochondria into cancer cells would inhibit proliferation. Finally we determined if addition of normal mitochondria increases the sensitivity of human breast cancer MCF-7 cells to chemotherapy. We found JC-1 stained mitochondria from normal mammary epithelial MCF-12A cells can enter into the cancer cell lines MCF-7, MDA-MB-231, and NCI/ADR-Res, but cannot enter normal MCF-12 A cells. The normal mitochondria from normal MCF-12 A cells suppressed the proliferation of MCF-7 and NCI/ADR-Res cells in a dose dependent pattern, but did not affect the proliferation of normal MCF-12A cells. The normal mitochondria from normal MCF-12A cells increased the sensitivity of human breast cancer MCF-7 cells to doxorubicin, Abraxane, and carboplatin. In conclusion the introduction of normal mammary mitochondria into human cancer cells inhibits cancer cell proliferation and increases the sensitivity of the MCF-7 human breast cancer cells to doxorubicin, Abraxane, and carboplatin. This phenomenon has never been reported, and the results support the role of mitochondrial dysfunction in cancer and suggest the possible use of targeted mitochondria as an adjunct to cancer therapeutics.

Citation Format: Robert L. Elliott, Xian P. Jian, Jonathan F. Head. Normal mammary epithelial mitochondria introduced into human breast cancer cells results in inhibition of proliferation and increased drug sensitivity of the MCF-7 breast cancer cell line. [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 5420. doi:10.1158/1538-7445.AM2013-5420
Abstract 5428: Rewiring of glucose metabolism in cancer.
Authors: Locasale Jason W.
Published: August 2014

This seminar topic focuses on the role glucose metabolism in cancer biology. A common theme within this biology is a rewiring of metabolism to support and drive these processes. I will discuss how changes in the metabolism of cancer cells alters the physic-chemical properties of the metabolic network. At the core of this effort lies the utilization of computational modeling and mass spectrometry. Using these tools, we have studied the effects of an alternative glycolytic pathway observed in proliferating cells that we recently identified. These efforts have enumerated several principles of glycolytic regulation in growing cells. We have also defined and characterized a metabolic pathway that is genetically selected for and drives the development of human cancer. This pathway involves the diversion of glycolytic flux into de novo serine metabolism through phosphoglycerate dehydrogenase (PHGDH). This direction has also led us to the study of how metabolism can regulate pluripotency and cell fate determination. Together, these findings define a systems biology approach through combining these mathematical tools with an integration of genetics, biochemistry, cell biology, and metabolomics.

Citation Format: Jason W. Locasale. Rewiring of glucose metabolism in cancer. [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 5428. doi:10.1158/1538-7445.AM2013-5428
Abstract 5430: Docosahexaenoic acid attenuates glycolytic metabolism and the Warburg phenotype by targeting hypoxia inducible factor 1 alpha in breast cancer.
Authors: Mouradian Michael , Kikawa Keith D. , Pardini Ronald S.
Published: August 2014

Dietary factors have been estimated to account for approximately 30-40 percent of cancer incidence and research has shown that high fat diets rich in n-3 polyunsaturated fatty acids (PUFAs) are associated with lower cancer occurrences, including breast cancer. The glycolytic switch, known as the Warburg Effect, is observed in many malignant models, which results in a unique metabolic shift in energy metabolism where tumor cells increase anaerobic glycolysis even in the presence of normal oxygen levels. In our laboratory, we have shown that the n-3 PUFA, docosahexaenoic acid (DHA; C22:6), significantly decreases total cellular ATP and the transcriptional activity and protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), a major contributor to the Warburg phenotype. In this project, we set out to determine if DHA is responsible for attenuating the Warburg Effect through a HIF-1α-dependent mechanism. Alterations in glucose uptake and usage upon supplementation with DHA were examined in vitro in the breast cancer BT-474, MDA-MB-231, and non-transformed MCF-10A cell lines. Total glucose transporter 1 (GLUT 1) protein levels were determined in each cell line. No change was seen upon DHA supplementation in the MDA-MB-231 cell line, which may be due to overexpression of GLUT 1. However, GLUT 1 protein levels were significantly down in response to DHA treatment in the BT-474 cell line. We also observed significant decreases in glucose consumption through glycolysis and lactate production in the BT-474 and MDA-MB-231 cell lines upon DHA supplementation. However, no change was seen in the non-transformed cell line MCF-10A, suggesting that DHA is having anti-glucose-metabolism effects specifically on cancer cells, but not on non-transformed cells. Moreover, HIF-1α small interfering RNA (siRNA) experiments showed a similar trend in GLUT 1 expression and glycolytic capacity in the BT-474 and MDA-MB-231 cell lines, suggesting that DHA is exerting some of its anti-cancer effects through HIF-1α. Based on these findings, changes in glucose transporter expression and glucose usage likely contribute to the impaired growth and survival of malignant cell lines supplemented with DHA. Currently, therapies are being developed to target the Warburg Effect in the treatment of cancer and DHA could likely enhance such therapies.

Citation Format: Michael Mouradian, Keith D. Kikawa, Ronald S. Pardini. Docosahexaenoic acid attenuates glycolytic metabolism and the Warburg phenotype by targeting hypoxia inducible factor 1 alpha in breast cancer. [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 5430. doi:10.1158/1538-7445.AM2013-5430
Abstract 5440: Analyzing in vivo conformational changes of a transcription factor, c-Myb, using fluorescence resonance energy transfer (FRET) technology.
Authors: George Olivia L. , Ness Scott A.
Published: August 2014

The c-myb proto-oncogene was first identified as the normal cellular counterpart to the v-myb oncogene identified in Avian Myeloblastosis virus (AMV), a retrovirus that transforms immature hematopoietic cells in tissue culture and induces leukemias in animals. However, the normal c-Myb protein is not oncogenic, in part because of conformational and auto-inhibitory interactions that keep its transforming activity in check. The c-Myb protein functions as both an activator and repressor of transcription and its regulation involves sequences found in the negative regulatory domain. We hypothesize that c-Myb protein undergoes conformational changes due to post-translational modifications and protein interactions that may help direct it to different sets of target gene promoters. In addition, there could also be upstream signaling pathways, for example the signals that direct cells either to proliferate or to undergo differentiation, that could contribute to its transforming potential. To address this, we are utilizing FRET technology to elucidate whether the intramolecular interactions of c-Myb or its interactions with various co-factors and co-activators change in vivo in response to extracellular signals that trigger a switch from proliferation to differentiation. The goal is to determine how the conformations and protein-protein interactions of c-Myb change in cells in response to signals that control cell fate.

Citation Format: Olivia L. George, Scott A. Ness. Analyzing in vivo conformational changes of a transcription factor, c-Myb, using fluorescence resonance energy transfer (FRET) technology. [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 5440. doi:10.1158/1538-7445.AM2013-5440
Abstract 5441: The involvement of MSK1 in non-canonical activation of NF-KB in the late phase of UVB irradiation.
Authors: Carpenter Oliver L.
Published: August 2014

Nuclear Factor Kappa-B (NF-KB) is a transcription factor that plays a crucial role in controlling expression of genes involved in the immune and inflammatory responses as well a being a key component in the onset of a larger number of cancers. One activator of NF-KB that has been studied, but still remains controversial, is that of ultraviolet B light (UVB). In this study we demonstrate that mitogen- and-stress activated protein kinase (MSK1) is responsible for a non-canonical late-phase activation of NF-KB. Following UVB irradiation (50 mJ/cm2) MSK1 is activated via phosphorylation at both 6 and 24 h time points. Investigations into the signaling pathways upstream of MSK1 through the use of specific inhibitors for mitogen-activated protein kinase kinase (MEK)(PD 98059) and p38 (SB 203580) revealed that both kinases are required for full phosphorylation during the late-phase (24 h) while p38 is predominately responsible for phosphorylation during the early-phase (6 h). Electromobilty shift assays (EMSA) for NF-KB binding affinity showed that inhibition of MSK1 via inhibitor H89 resulted in a marked reduction in binding affinity. Inhibition of p38 also coincided with a reduction in NF-KB binding affinity. Interestingly, supershift assays implicate that the p65 isoform of NF-KB is predominantly involved in late-phase activation in response to UVB irradiation. Analysis of cell viability showed that decreased viability was correlated with MSK1 inhibition and decreased NF-KB binding affinity. Together, the results of these studies shed light onto a novel pathway of MSK1 mediated late-phase activation of NF-KB in response to UVB irradiation.

Citation Format: Oliver L. Carpenter. The involvement of MSK1 in non-canonical activation of NF-KB in the late phase of UVB irradiation. [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 5441. doi:10.1158/1538-7445.AM2013-5441
Abstract 5442: The role of ATF3 in arsenic-induced GADD45 expression.
Authors: Shi Qiwen , Bhatia Deepak , Sutariya Vijaykumar B.
Published: August 2014

GADD45 (growth arrest and DNA damage inducible gene 45), an effector of G2/M checkpoint, is induced by trivalent arsenic (As3+) in human bronchial epithelial cell line, BEAS-2B. In the present study, we investigated the regulation of ATF3 (activating transcription factor 3) on GADD45 with As 3+ treatment. We demonstrated that As3+ induced ATF3 expression, at both mRNA and protein level, and the induction was reduced by p38 and JNK inhibition, but not affected by PI3K and ERK inhibition. Luciferase assay also confirmed As3+ involvement in ATF3 transcriptional regulation. Moreover, silencing of ATF3 downregulated As3+-induced expression of GADD45 and chromatin immunoprecipitation suggested that ATF3 acted as an activator of GADD45 transcription in the presence of As3+. Taken together, our data indicated that the increase of GADD45 in response to As3+ was mediated by ATF3.

Citation Format: Qiwen Shi, Deepak Bhatia, Vijaykumar B. Sutariya. The role of ATF3 in arsenic-induced GADD45 expression. [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 5442. doi:10.1158/1538-7445.AM2013-5442