bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2013: Modulation of cellular uptake and increased therapeutic efficacy of the azacytidine-elaidic acid ester CP-4200 in vitro and in vivo
Authors: Brueckner Bodo , Rius Maria , Markelova Maria Rivera , Fichtner Iduna , Hals Petter-Arnt , Sandvold Marit Liland , Lyko Frank
Published: July 2012

Azacytidine is an established nucleoside drug that is well known for its ability to modulate epigenetic gene regulation by inhibition of DNA methylation. Despite recent advances in the clinical development of azacytidine, the use of the drug is limited by its low bioavailability and dependency on variably expressed nucleoside transporters for cellular uptake. To investigate the function of CP-4200, an elaidic acid ester of azacytidine, we performed DNA methyltransferase depletion assays, DNA methylation analysis using capillary electrophoresis and Infinium methylation arrays, transport studies and mouse experiments. We show here that CP-4200 has strong epigenetic modulatory potency in human cancer cell lines, as evidenced by efficient depletion of DNA methyltransferase protein, genome-wide DNA demethylation and robust reactivation of epigenetically silenced tumor suppressor genes. Importantly, new results show that the uptake mechanisms of colon and leukemic cancer cell lines for CP-4200 and its parent drug azacytidine are fundamentally different, with CP-4200 being substantially less dependent on nucleoside transporters. Correspondingly, CP-4200 showed a significantly higher antitumoral activity than azacytidine in an orthotopic mouse tumor model for acute lymphocytic leukemia. These results establish a fundamentally novel approach for the delivery of azacytidine to human cancer cells which might have important therapeutic implications.

Reference: Brueckner, B., et al. (2010). Mol. Cancer Ther. 9: 1256-1264.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2013. doi:10.1158/1538-7445.AM2011-2013
Abstract 2015: Effect of valproic acid on fludarabine activity in chronic lymphocytic leukemia patients
Authors: Yoon Ju-Yoon , Szwajcer David , Ganchimeg Ishdorj , Benjaminson Pat , Johnston James , Gibson Spencer
Published: July 2012

Valproic acid (VPA) is a first-generation anti-epileptic drug that has been shown to inhibit proliferation and induce apoptosis in various hematological malignancies, including multiple myeloma and chronic lymphocytic leukemia (CLL). We show here that the mechanism of VPA-induced apoptosis in CLL is associated with activation of the TNF-related apoptosis-inducing ligand (TRAIL) apoptotic pathway in vitro and in vivo. Treatment of leukemic cell lines or primary CLL cells with VPA increased the level of TRAIL receptor 1 (TRAIL-R1), but not TRAIL-R2, and upregulated the TRAIL pathway in vitro, resulting in increased casepase-8 cleavage. Treatment with VPA also induced formation of reactive oxygen species (ROS), and the combined cytotoxicity of VPA and fludarabine was dampened upon co-treatment with a ROS scavenger, N-acetylcysteine. Having shown the effects of VPA in vitro, a phase II clinical trial was initiated at CancerCare Manitoba to determine the activity of VPA in CLL, either when used alone or in combination with fludarabine. Five patients who had received at least one prior therapy with fludarabine have to date been examined. Three out of 5 patients were fludarabine-resistant, as defined as no response to fludarabine or relapse <6 months after completion of treatment with fludarabine. No responses were seen after 28 days using VPA alone. However, in four patients who continued on VPA with fludarabine, 3 patients showed a >50% fall in lymphocyte/lymph node size after receiving 5 cycles of the combination. Peripheral blood samples were obtained prior to each treatment cycle, and mononuclear cells were isolated for analysis by immunoblotting and immunocytochemistry. Levels of both histone 3-acetyl and histone 4-acetyl initially increased and then fluctuated during the course of treatment. TRAIL-R1 levels increased during the course of therapy, while no significant changes in TRAIL-R2 levels were observed. Expression of mRNA levels of genes involved in apoptosis was examined in one patient before and 28 days following VPA. There was a global up-regulation of both pro- and anti-apoptotic genes (as categorized by their gene ontology) which likely explains the effect of VPA on fludarabine sensitivity in CLL. In summary, VPA induces hyper-acetylation of histones in CLL and activates the TRAIL apoptotic pathway. While VPA is ineffective as a single agent in CLL, it can sensitize CLL cells to fludarabine and could be used as an adjuvant in fludarabine-based treatment regimens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2015. doi:10.1158/1538-7445.AM2011-2015
Abstract 2018: Combination therapy with vidaza and entinostat suppresses tumor growth and reprograms the epigenome in an orthotopic lung cancer model
Authors: Li Nan , Grimes Marcie J. , Picchi Maria A. , Mitchell Hugh D. , Stidley Christine A. , Tesfaigzi Yohannes , Channell Meghan M. , Liu Yanbin , Casero Robert A. , Baylin Stephen B. , Reed Mathew D. , Tellez Carmen S. , March Thomas H. , Belinsky Steven A.
Published: July 2012

Epigenetic therapy for solid tumors could benefit from an in vivo model that defines tumor characteristics of responsiveness and resistance to facilitate patient selection. Here we report that combining the histone deacetylase inhibitor entinostat with the demethylating agent vidaza profoundly affected growth of K-ras/p53 mutant lung adenocarcinomas engrafted orthotopically in immunocompromised nude rats by targeting and ablating pleomorphic cells that occupied up to 75% of the tumor masses. A similar reduction in tumor burden was seen with epigenetic therapy in K-ras or EGFR mutant tumors growing orthotopically. Increased expression of pro-apoptotic genes and the cyclin dependent kinase inhibitor p21 was seen. Hundreds of genes were demethylated highlighted by the re-expression of polycomb-regulated genes coding for transcription factor binding proteins and the p16 gene, a key regulator of the cell cycle. Highly significant gene expression changes were seen in key regulatory pathways involved in cell cycle, DNA damage, apoptosis, and tissue remodeling. These findings demonstrate the promise for epigenetic therapy in cancer management and provide an orthotopic lung cancer model that can assess therapeutic efficacy and reprogramming of the epigenome in tumors harboring different genetic and epigenetic profiles to guide use of these drugs. This study was supported by the Waxman Foundation and by a research grant from NIH (R01 CA095568).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2018. doi:10.1158/1538-7445.AM2011-2018
Abstract 2022: Wnt ligands are overexpressed in murine mammary tumors in BK5.ATF3 transgenic mice
Authors: Yan Leqin , Powell K. Leslie , MacLeod Michael C.
Published: July 2012

The Wnt/β-catenin signaling pathway is up-regulated in mammary tumors that arise with 70% incidence in parous female BK5.ATF3 transgenic mice. Nuclear accumulation of β-catenin is seen in both basal and supra-basal tumor cells in this model, whereas nuclear expression of the transcription factor ATF3 is only seen in the basal cell layer of the tumors. If ATF3 acted as a positive regulator of one or more Wnt ligands in the basal cells, one might expect paracrine upregulation of the Wnt/β-catenin pathway in neighboring supra-basal cells. To test this, we measured mRNA expression levels for 9 Wnt genes in BK5.ATF3 induced mammary tumors and normal mammary glands. The genes for five known positive acting Wnt ligands, Wnt3, Wnt3a, Wnt5a, Wnt7b, and Wnt10b, were significantly upregulated in tumors as compared to transgenic mammary glands. One of these, Wnt3, was significantly down-regulated in non-transgenic glands. In a mouse mammary tumor cell line, EMT6, in which the endogenous Atf3 gene is overexpressed, the only Wnt gene for which expression could be detected by qPCR was Wnt7b. ChIP assays demonstrated binding of both Atf3 and Jun proteins to two putative AP-1 sites upstream of the Wnt7b transcription start site in these cells. ATF3 and JUN are known in other systems to form heterodimers that exhibit positive transactivation through AP-1 sites, and we have identified a physical interaction between ATF3 and JUN in BK5.ATF3-induced tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2022. doi:10.1158/1538-7445.AM2011-2022
Abstract 2033: Transcription factors that regulate MUC16 expression in ovarian cancer
Authors: Thapi Dharmarao , Binder Darcie , Rosales Nestor , Yan Xiu Jun , Lash Alex E. , Spriggs David R.
Published: July 2012

CA125 antigen is found to be elevated in 85% of patients diagnosed with advanced epithelial ovarian cancer. CA125 has a minimal role as a screening modality. The gene which encodes for the CA125 glycoprotein, MUC16, has been previously identified and sequenced. MUC16 has a molecular size of 22,000 bps and consists of cytoplasmic domain, transmembrane region and 9 external domain tandem repeats. The large molecular weight of this gene has made it difficult to study and track. N terminal signal peptide and transcription factor or factors that triggers MUC16 in ovarian cancer is not well demonstrated. The proximal 114 amino acids of MUC16 can transform NIH/3T3 cells and increase soft agar formation and matrigel invasion.

We have designed several constructs from the region about 1000 nucleotides upstream and 300 nucleotides downstream of the MUC16 start Methionine with secretory pMetridia Luciferase Reporter vector (Clonetech, CA). Four constructs of varying lengths had the MUC16 UTR TSS site whereas two did not. Putative binding sites for transcription factors like ER, STAT1, STAT3, CRE-BP1 (ATF2) and NFKB are also present. These constructs were then transfected individually into CA125 negative ovarian cell line, SK-Ov-3, and also into CA125 positive ovarian cell lines, SK-Ov-8 and CAOV3. All cell lines were stably selected with G418 and were used within 10 passages. Western blots confirm the presence of ERalpha, STAT1 and STAT3.

Stably transfected cell lines were cultured in low serum conditions with or without 17β-Estradiol or IL-1β, were used to see the effect of transcription. Clinical CA125 levels were also documented for each construct. Secretory pMetridia Luciferase signals were detected in the supernant of the culture cells. There is no difference between untreated or 17β-Estradiol or IL-1β treated cells. However, gel retardation studies with OVCAR3 and SKOV8 wild type cell lines suggested CREB/ATF (CREBZF) transcription factor involvement and confirmatory reporter gene studies are planned. Currently, we are assessing the putative transcription factor binding sites within the 1300 nucleotides region from which we derived the constructs.

We conclude that these studies will lead us to better understand the biology of MUC16 gene expression and give us an insight about the role of transcription factors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2033. doi:10.1158/1538-7445.AM2011-2033
Abstract 2050: 2-Deoxy-D-Glucose (2-DG) as a glycolytic inhibitor in the treatment of retinoblastoma
Authors: Decatur Christina , Pina Yolanda , Houston Samuel , Cavalcante Ludimila , Shah Nikesh , Murray Timothy
Published: July 2012

Purpose: The aim of the current study is to assess the impact of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on tumor burden and hypoxia in the LHBETATAG retinal tumors.

Methods: Group A: 17-week-old LHBETATAG transgenic mice (n=30) received periocular injections of saline and 2-DG (62.5, 125, 250, and 500mg/kg). Injections were given 2 times a week for 3 weeks. Group B: 4-week-old mice received oral delivery of 2-DG in custom made food pellets and were treated for either 8 or 18 weeks. 17-week-old mice received oral 2-DG for 8 weeks. At the time of enucleation, all eye samples were snap frozen and analyzed for tumor burden and hypoxic regions using immunohistochemistry.

Results: Following injections of 2-DG, tumor control was not different between the control and the lowest two doses (62.5 and 125mg/kg). However, the difference in tumor burden was significant from 250mg/kg dose (p<0.015) and 500mg/kg dose (p<0.001) with 9.7 and 23% tumor burden decrease, respectively. At all doses of periocular 2-DG, the percent hypoxia following drug treatment were lower relative to controls (p<0.001), with a hypoxia decrease in the lowest dose to 2.4% compared to 21% in controls. Following oral delivery of 2-DG, there was a difference between the groups treated with 2-DG and the control groups (p<0.010). A 20% reduction in tumor burden was found in the 8 weeks early treated, a 50% reduction in the 18 weeks early treated, and a 35% reduction in the 8 weeks late treated group. The percent hypoxia was lower relative to controls in the 8 weeks and 18 weeks early treated groups (p<0.001) with a 40% and 50% reduction, respectively. The percent hypoxia did not change following treatment in the treated late group.

Conclusions: This study exhibits the efficacy of focal 2-DG as potential therapy to decrease both intratumoral hypoxia and tumor burden. The current study also shows that the non-invasive oral delivery 2-DG treatment has similar or better effects on the tumor depending on the treatment schedule used. In advanced disease of LHBETATAG retinal tumors, hypoxia is increasingly present. The use of glycolytic inhibitors as a therapeutic strategy has the potential to enhance current retinoblastoma treatments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2011-2050
Abstract 2056: Evidence against hypoxic-dependent activation of carbonic anhydrase IX in MDA-MB-231 breast cancer cells
Authors: Frost Susan C. , Li Ying , Tu Chingkuang , Silverman David N.
Published: July 2012

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme the expression of which is often considered a marker for hypoxia, an indicator of poor prognosis, and associated with acidification of the tumor microenvironment. Many studies have shown that CAIX expression is induced by hypoxia exposing its catalytic domain to the interstitial milieu. Several recent studies have suggested that hypoxic conditions may also permit activation of CAIX, perhaps by causing a conformational change which exposes the catalytic pocket. Our goal was to assess the effect of anoxic conditions on CAIX activity in MDA-MB-231 cells, previously exposed to hypoxia which increases CAIX expression in the absence of other membrane-bound carbonic anhydrase (CA) family members.

We have taken advantage of membrane inlet mass spectrometry (MIMS) to directly analyze CA activity in intact cells by measuring the 18O exchange between CO2 and H2O. This method distinguishes between intracellular and extracellular CA activity. MDA-MB-231 cells were exposed to 1% oxygen for 16 hours after which they were isolated under normoxic or anoxic conditions. CA activity was then measured, again under normoxic or anoxic conditions. These data show biphasic depletion of 18O from CO2 under both normoxic and anoxic assay conditions. The first phase (which occurs over the first 20-40 seconds) represents a rapid diffusion of CO2 into cells where it is exposed to intracellular CAII. A catalytic cycle depletes 18O followed by efflux of CO2 from the cell. There was no difference in this phase between cells prepared and assayed under normoxic or anoxic conditions. The second phase (from 200-500 sec) is dominated by the hydration-dehydration reaction of CO2/HCO3− catalyzed by exofacial CA activity (CAIX). In cells exposed to anoxia, the slope of the second phase was greater than that observed with cells exposed to normoxic conditions indicating elevated CAIX activity. While this provided evidence that oxygen limitation might influence CAIX activity, the Ki values for two impermeant CA inhibitors, Cpd 5C and a polymeric sulfonamide, did not differ between anoxic and normoxic cells. We conclude from these data that the catalytic site of CAIX is exposed and functional under both anoxic or normoxic conditions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2011-2056
Abstract 2070: XBP-1 promotes cell survival by activating the unfolded protein response (UPR) in antiestrogen resistance in breast cancer
Authors: Shajahan Ayesha N. , Hickman F. Edward , Cook Katherine , Clarke Robert
Published: July 2012

Most of the 192,000 newly diagnosed cases of invasive breast cancer in 2011 will be estrogen receptor positive alpha positive (ER+). Endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole (LET), is the least toxic and most effective means to manage the hormone-dependent breast cancer in such patients. However, resistance to endocrine therapy remains a significant clinical problem. Our group has shown that antiestrogen resistant breast cancer cells over-express X-Box Binding Protein 1 (XBP1), a transcription factor that belongs to the basic region/leucine zipper (bZIP) family. XBP1(U) and XBP1(S) variants result from an unconventional splicing of the XBP1 mRNA by IRE1alpha. In the unfolded protein response (UPR), endoplasmic reticulum proteins (IRE1alpha, ATF6 and PERK) sense cellular stress to regulate the accumulation of unfolded proteins. Initially a compensatory mechanism allowing cells to recover normal endoplasmic reticulum function, prolonged UPR may induce cell death; this is often dependent upon which arm of the UPR predominates. XBP1 is an obligate component in both the IRE1alpha and ATF6 arms of the UPR. Our study shows that antiestrogen resistant breast cancer cells survive by activating pro-survival autophagy through XBP1-mediated UPR. MCF7/LCC9 [Faslodex resistant; TAM cross-resistant; high level of XBP1(S)] cells showed an increase in UPR signaling as detected by increased expression of BiP/GRP78. Antiestrogen-sensitive cells that over-express XBP1(S) show decreased sensitivity to Faslodex and show increased levels of basal and Faslodex-induced autophagy as measured by cleaved LC3BII protein fragment, GFP-LC3 activity, and reduced expression of p62/SQSTM1. Thus, we conclude that antiestrogen resistant breast cancer cells evade cell death by activating the XBP1-regulated arms of the UPR that results in prosurvival autophagy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2070. doi:10.1158/1538-7445.AM2011-2070
Abstract 2075: miRs are altered by caloric restriction: Therapeutic implications
Authors: Saleh Anthony , Savage Jason E. , Harris Curtis C. , Mitchell James B. , Simone Nicole L.
Published: July 2012

The anti-cancer effect of caloric restriction (CR) is thought to involve repression of insulin/IGF-1 signaling, which activates PI3k/AKT, a pro-proliferation pathway that is often up regulated in tumor cells. Several microRNAs have been shown to play roles in tumor suppression and oncogenesis. In this study we sought to determine what if any microRNAs can play a role in the anticancer effects of caloric restriction. To determine this we performed microRNA arrays on total RNA from ad libitum fed, CR, or 24-hour fasted C57b/J mice. We assessed liver and brain tissues since these are known to have great alterations with caloric restriction and we also assessed breast tissue for which there is little data with dietary alteration. Our results show that both caloric restriction and fasting alters the expression of several cancer relevant microRNAs all of the tissues analyzed. A reduction of the miR-200 and mir-17-92 cluster of miRNAs was noted and these miR's are known to be upregulated in many cancers. Conversely, increases in the mir-29 and mir-30 families of miRNAs were noted which are noted to be downregulated in many cancers. mir-29 is known to target p85 protein of PI3K which would also reduce AKT signaling. In addition, mir-29 and mir-30 are predicted to target IGF-1 and IGF-1R respectively. Analyses of protein and mRNA levels confirm that IGF-1 and IGF-1 R are down regulated by CR and fasting. Cell culture studies are ongoing to show direct targeting of these transcripts by mir-29 and mir-30. This study shows that microRNAs are involved in the potential benefits of caloric restriction and show a general role for regulation of insulin related signaling by mir-29 and mir-30 which may be provide a target that can be exploited for therapeutic benefit.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2075. doi:10.1158/1538-7445.AM2011-2075
Abstract 2085: Reactive oxygen species promote signaling in neuroblastoma cells
Authors: Harrison Dominick , Joanis Ruth , van Golen Cynthia M.
Published: July 2012

Neuroblastoma is a rare cancer of the sympathetic nervous system that is commonly diagnosed in children before the age of five. Neuroblastoma affects 1 out of 100,000 children and about 700 new cases of neuroblastoma each year. Most cases of neuroblastoma begin in the abdomen in the adrenal gland, or next to spinal cord and in the chest. Neuroblastoma is known to spread to the bones, face, skull, pelvis, shoulder, arms, legs, bone marrow, liver, lymph nodes, skin and around the eyes. Currently, there is no explanation for the cause of neuroblastoma. Our lab seeks to understand the mechanisms used for these cells to metastasize. The Erk pathway, part of the mitogen activated protein kinases, helps to regulate the cell cycle, proliferation, invasion and metastasis. The PI3k/Akt pathway regulates cell growth, survival, and activation of transcription factors such as hypoxia inducible factors (HIFs). Hypoxia is a state where the cell is experiencing a lower than normal oxygen tension activating HIF-1 alpha and HIF-2 to cause an expression of target genes for angiogenesis, (generation of new blood cells), erythropoiesis (formation of new blood cells), and glucose transport into cells enabling them to survive under hypoxic conditions and acquire access to an oxygen supply. Hypoxia increases reactive oxygen species used to stabilize HIF-1 alpha. Reactive oxygen species generation is dependent on oxygen induced changes in the redox state of the electron transport chain. Low levels of reactive oxygen species can regulate controlled proliferation. However, high levels of reactive oxygen species lead to DNA damage and genomic instability by activating these signaling pathways stopping cell death in senescence leading to uncontrolled proliferation. Therefore, our hypothesis is that low levels of reactive oxygen species promote neuroblastoma cell survival through upregulation of survival signaling, whereas high levels of reactive oxygen species induce neuroblastoma cell apoptosis. To test this idea, we exposed an aggressive, metastatic cell lines, IMR 32 neuroblastoma cells, to increasing concentrations of hydrogen peroxide, used to induce reactive oxygen species formation. Cells were analyzed morphologically for apoptosis. With increasing concentrations of hydrogen peroxide, IMR32 neuroblastoma cells round up and become less adherent. High doses of hydrogen peroxide induce cell fragmentation indicative of apoptosis. We are currently investigating the effect of hydrogen peroxide on the induction of caspase-3, a proapoptotic molecule, and DNA fragmentation, measured by TUNEL assay. Our data suggest that reactive oxygen species induce apoptosis in human neuroblastoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2085. doi:10.1158/1538-7445.AM2011-2085
Abstract 2095: Tunicamycin inhibits triple negative breast cancer progression
Authors: Banerjee Aditi , Duran Elizabeth , Couto Javier A. , Zhang Zhenbo , Espinoza Lydia A. , Banerjee Sushanta K. , Baksi Krishna , Banerjee Dipak K.
Published: July 2012

Triple negative (ER−/PR−/EGFR−) breast cancer accounts for 15% of all breast cancers. A large percentage of them are African and African-American women who are premenopausal. Histologically, the cancers are poorly differentiated, and most fall into the basal subgroup of breast cancers. Because of the absence of specific treatment guidelines, these patients are managed with standard treatment which leaves them associated with a high rate of local and systemic relapse. Angiogenesis is crucial for breast tumor growth and progression. Our laboratory has recently established that Tunicamycin (TM; an antibiotic and a glucosamine-containing pyrimidine nucleoside) interferes with the dynamic relationship between asparagine-linked (N-linked) glycosylation and angiogenesis. As a result, the xenograft developed in athymic nude mice [Balb/c (nu/nu)] with human breast cancer cell line MDA-MB-231 is reduced ∼65% in one week when Tunicamycin was given orally (0.25 mg/Kg) twice per week. To understand if it is due to a direct effect of Tunicamycin on the tumor microvsculature or a nutritional deprivation of the tumor cells due to insufficient blood flow, we have studied the effect of Tunicamycin on MDA-MB-231 breast cancer cells. When titrated with Tunicamycin (0.1 µg/ml, 1.0 µg/ml and 10.0 µg/ml) for seven days, the cell growth was inhibited almost quantitatively at all concentrations and as a function of time. In addition, no colony was formed at 1 µg/ml of Tunicamycin, the concentration used in all our experiments. Protein expression by western blotting and the gene expression by qRT-PCR indicated down-regulation of cyclin D1, and up-regulation of wild type p53, phopspho p53(ser 392), Bcl-2 and caspase-3 with no changes in the cdk-4 expression. These results strongly supported cell cycle arrest in G1 followed by the induction of apoptosis. Our recent study has demonstrated that Tunicamycin induces ER stress and consequently the apoptosis by unfolded protein response (upr) in breast tumor microvasculature. When examined, the MDA-MB-231 cells expressed high levels of GRP-78 as well as phospho PERK, a translational attenuator. Thus, we conclude that the MDA-MB-231 breast cancer cells experienced upr-mediated apoptotic death in the presence of Tunicamycin and strongly supported Tunicamycin's potential for a glycotherapeutic for treating a triple negative breast cancer in the clinic. Supported in part by grants from Susan G. Komen for the Cure (BCTR06582) and NIH/NCRR/RCMI G12-RR03035 (KB).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2095. doi:10.1158/1538-7445.AM2011-2095
Abstract 2096: Estrogen receptor alpha activation mediates a novel mitochondrial unfolded protein response
Authors: Papa Luena , Germain Doris
Published: July 2012

Unfolded protein responses (UPR) of the endoplasmic-reticulum and of the matrix of the mitochondria have been described. Our data indicates that accumulation of proteins in the inter-membrane space of the mitochondria (IMS) activates a distinct UPR from that induced by stress in the mitochondrial matrix. We show that upon IMS-stress, cells activate the Estrogen Receptor (ERα) in a ligand independent fashion through ROS overproduction and AKT phosphorylation. ERα activation further triggers the induction of the mitochondrial regulator NRF-1 and the IMS protease Omi. In addition, IMS stress leads to an acceleration in the trypsin-like activity of the proteasome. Since we reported a proteasome and Omi-dependent checkpoint that limits the import of IMS proteins, these results suggest that this novel UPR acts as cyto-protective responses to overcome IMS-stress. Indeed, we show that whereas ERα is required for activation of these cyto-protective responses, its ligand-independent activation is necessary to prevent IMS stress-induced collapse in the mitochondrial potential. Notably, our result show that upon mitochondrial dysfunction, cells trigger the retrograde pathway involving the activation of ERα to facilitate the recovery of the physiological functions of the mitochondria and maintain their integrity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2011-2096
Abstract 2103: Reduced Tip60 expression as a predictive biomarker for advanced melanoma and patient outcome
Authors: Chen Guangdi , Chen Yabin , Zhang Zhizhong , Martinka Magdalena , Li Gang
Published: July 2012

The tumor suppressor Tip60 plays a major role in transcription, DNA damage response, apoptosis and cancer development. We investigated the role of Tip60 in melanoma pathogenesis and assessed the prognostic value of Tip60 expression on melanoma patient survival. Two sets of tissue microarrays were constructed consisting of 448 cases of melanomas (201 for the training set and 247 for the validation set) and 105 cases of nevi. The TMA was assessed for Tip60 expression by immunohistochemistry. The Kaplan-Meier method was used to evaluate the patient survival, and univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up. We found that Tip60 expression was significantly reduced in metastatic melanoma compared to normal nevi (P = 0.045), dysplastic nevi (P = 0.047) and primary melanoma (P = 0.001). Tip60 expression correlated with AJCC stages (I-II vs III-IV, P = 0.001) and tumor thickness (≤4.00 vs. >4.00 mm, P = 0.037). Reduced Tip60 expression was associated with a poor five-year disease-specific survival in primary melanoma (P = 0.016) and metastatic melanoma patients (P = 0.027). Furthermore, Cox regression analyses indicated that Tip60 expression was an independent prognostic marker for primary (P = 0.002) and metastatic melanomas (P = 0.035). In conclusion, reduced Tip60 expression is significantly associated with thick primary melanoma and metastatic melanoma, as well as poor patient prognosis, suggesting that Tip60 may serve as a potential biomarker for advanced melanoma and patient outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2103. doi:10.1158/1538-7445.AM2011-2103
Abstract 2109: Tumor progression locus 2 (Tpl2) knockout mice have a dysregulation in COX-2 signaling in two-stage skin carcinogenesis
Authors: DeCicco-Skinner Kathleen L. , Nolan Sabrina , Dempsey Taylor , Trovato Erika , Weist Jonathan
Published: July 2012

Skin cancer is the most common form of cancer in the United States, with an estimated two million cases diagnosed annually. Inflammation is believed to be a critical component in skin cancer progression, and therefore understanding genes controlling inflammation is beneficial. MAP3K8 (Tpl2) is a protein kinase in the MAP Kinase signal transduction cascade. Previous research using a two-stage skin carcinogenesis model has revealed that Tpl2 -/- mice have significantly higher tumor incidence and inflammation than wild-type (WT) controls. Mechanistically, dysregulation in NF-κB signaling is a major contributor to the high degree of inflammation and tumorigenesis found in the Tpl2 -/- mice. Knockout animals have heightened NF-κB reporter activity, increased NF-κB dependent edema, and increased NF-κB nuclear localization. This study investigates how cyclooxygenase-2 (COX-2), an NF-κB regulated gene known to contribute to skin carcinogenesis, and COX-2 downstream factors might play a role in inflammation-mediated skin carcinogenesis in Tpl2 -/- mice. Keratinocytes from newborn WT or Tpl2 -/- mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for varying times over 24 hours. Western analysis revealed significant differences in COX-2 and COX-2 dependent prostanoids and prostanoid receptors. Additionally, in vivo experiments confirmed that COX-2 and COX-2 downstream factors were elevated in TPA-treated Tpl2 -/- skin, as well as in papillomas and squamous cell carcinomas from Tpl2-/- mice. These experiments illustrate that COX-2 induction in the absence of Tpl2 may be responsible for the increased tumorigenesis found in Tpl2-/- mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2109. doi:10.1158/1538-7445.AM2011-2109
Abstract 211: TIS21/BTG2/PC3 triggers cancer cell death, instead of cellular senescence, by enhancing proapoptotic gene expression at the downstream of p53
Authors: Devanand Preethi , Choi Ok Ran , Park Tae Jun , Lim In Kyoung
Published: July 2012

It has been known that cytoplasmic p53 regulates cell cycle arrest, apoptosis, autophage, and metabolism of human and animal cells in a transactivation-dependent manner. Wild type p53 induces senescence of EJ human bladder cancer cells, which carries oncogenic H-ras and mutant p53. Tumor supporessor, TIS21/BTG2/PC3, is one of the target genes regulated by wild type p53. Employing adenoviral vectors carrying p53(Ad-p53) and TIS21(Ad-TIS21) genes, we investigated effect of TIS21 on cancer cell senescence induced by p53 supplementation; Infection of EJ cells with p53 rapidly induced senescence phenotypes, such as cellular enlargement, inhibition of its growth, expressions of SA-[[Unsupported Character – Symbol Font ]]-galactosidase and SA-pErk1/2, and generation of reactive oxygen species, in addition to down-regulation of PARP and pRb expressions. A mechanism of the senescence was likely to be upregulations of H-ras and paxillin expressions by p53, evidenced by employing si-p53 RNA construct. On the contrary, the combined effect of p53 plus TIS21 significantly induced apoptosis of EJ cells rather than senescence, when evaluated by Annexin V expression, Calcein-AM and EhtD-1 double staining, increased expressions of proapoptotic genes and caspase 3 activity, whereas TIS21 alone failed to induce any changes, as compared with those of the control. p53 plus TIS21 significantly increased Bax and Apaf-1 expressions, whereas paxillin expression was clearly reduced as compared with those of p53 alone. When its mechanism was investigated, acetylation of p53 on K120 and K373 residues were found to be increased in addition to the significant translocation of p53 after coexpression of TIS21. We strongly suggest here that TIS21 is an endogenous cell death promoting gene at the downstream of p53 even in the expressions of oncogenic H-Ras and mutant p53.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2011-211
Abstract 2111: Expression pattern and mechanism of tumor suppression of Rab25
Authors: Cheng Jiming , Ayyagari Vijayalakshmi , Sunkara Bharat , Koirala Sajan , Brahmamdam Abhinav , Rao Krishna A.
Published: July 2012

Introduction: Our previous work has shown that the loss of Rab25 occurs in estrogen receptor (ER) and progesterone receptor (PR) negative breast cancer cell lines and human tumor samples and seems to correlate with an active ras pathway. We have examined the expression levels of rab25 in breast tissue and have investigated the mechanism by which Rab25 exerts its anti-tumorigenic effect. Loss of Rab25 cooperates with mutant Ras to promote tumorigenesis. HIF-1α levels are reduced in Rab25 expressing cells. We have assessed Rab25 binding to the HIF-1α promoter and localized Rab25 to the nucleus, suggesting a novel mechanism for Rab25's antitumor activity and a novel function for Rab25.

Materials and Methods: Cell culture – Normal mammary tissue and the RAO-3 cell line was grown in DFCI-1 medium. Cells were transduced with retroviral supernatant and selected. Expression of Rab25 in transduced cells was determined by RT-PCR and western blot. qPCR analysis- Expression of HIF-1α and Rab25 were quantified using real-time PCR with fluorescence detection. Luciferase report assay – We obtained the HIF-1α promoter fragment and inserted the promoter sequence in the pGL4 luciferase vector and the Luciferase Assay was then performed. Western blot- Protein extracts from both cytosolic and nuclear fraction were tested for Rab25 expression. Immunofluorescence- Frozen mammary tissue slides were stained with luminal and basal markers as well as antibody for Rab25 to confirm localization of Rab25 in both luminal and basal mammary epithelium.

Results:

1. High level expression of Rab25 was found in luminal epithelium in normal human breast tissue. Basal epithelium also expressed Rab25.

2. Tumorigenic mammary epithelial cell lines, RAO-3 and RAO-4, have lost Rab 25 expression with nearly 1/10,000 the level of Rab25 of normal mammary basal epithelium.

3. Rab25 localizes to the nucleus and reduces luciferase activity from the HIF-1α promoter.

4. Expression of Rab25 substantially reduces HIF-1α levels.

Conclusions:

1. Rab25 is a complex molecule that has been described as an intracellular transport protein.

2. It is also a tumor modulator. It can regulate tumor growth and angiogenesis.

3. Rab25 expression can be detected in both luminal epithelium and basal epithelium.

4. Our data indicate novel functions for Rab25 including reduction in HIF-1α expression and localization to the nucleus.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2111. doi:10.1158/1538-7445.AM2011-2111
Abstract 2119: Role of annexin A6 in anchorage-independent growth of invasive breast cancer cells
Authors: Sakwe Amos M. , Koumangoye Rainelli , Ochieng Josiah
Published: July 2012

Annexin A6 (AnxA6) is a member of the annexin family of Ca2+-dependent membrane binding proteins that is frequently detected in cell-derived exosomes and on the surface of some cell types. Although its extracellular functions remain unclear, AnxA6 may play important roles in cell-cell and/or cell-extracellular matrix (ECM) interactions via its interactions with chondroitin sulfate containing proteoglycans, the serum glycoprotein fetuin-A and other undefined extracellular matrix components. In support of this notion, recent studies have shown that depletion of AnxA6 in MDA-MB-436 breast cancer cells promoted anchorage-independent cell growth. Here we show that depletion of AnxA6 in BT-549 breast carcinoma cells not only led to impaired cell-cell cohesion and cell spreading onto collagen type IV but also inhibited cell motility and invasiveness. In 3D cultures in growth factor-reduced matrigel, AnxA6-depleted BT-549 cells formed spheroid colonies as opposed to stellate colonies formed by the parental BT-549 cells. We also show that N-cadherin is up-regulated in the motility-impaired AnxA6-depleted cells, suggesting that loss of AnxA6 may uncouple cadherin-based cell-cell interactions as part of the mechanism that determines anchorage-independent cell growth and therefore tumor growth in vivo. Our data also show that the loss of contact inhibition and invasive properties in AnxA6-depleted cells may be partly due to the formation of fewer, elongated and dispersed vinculin-based focal contacts. To verify that these focal contacts may be functionally defective, we show that whereas the MAP kinase (ERK1/2) pathway remained constitutively active, Ca2+-dependent activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited in the AnxA6-depleted BT-549 cells. These data suggest that loss of AnxA6 may promote breast cancer progression via anchorage-independent cell growth due to disruption of functional focal contacts that maintain cell-cell and/or cell-ECM interactions. Supported by grants: 1 SC1 CA134018-01 and DOD W81XWH-07-1-0254

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2119. doi:10.1158/1538-7445.AM2011-2119
Abstract 2123: Cytoplasmic STAT1 expression level opposes disease-specific survival of sarcoma patients
Authors: Zimmerman Mary A. , Liu Kebin , Lev Dina C.
Published: July 2012

Signal Transducer and Activator of Transcription 1 (STAT1) is a key mediator in the interferon gamma (IFN-γ) signaling pathway. It is well known that IFN-γ-activated STAT1 (pSTAT1) plays a critical role in the suppression of tumor development. However, STAT1 has also been shown to act as a tumor promoter in certain types of cancers. These findings indicate that STAT1 harbors dual functionality, functioning as either a tumor suppressor, or a promoter, a distinction that may ultimately depend on the tumor microenvironment. In this study, we hypothesized that the distribution of STAT1 (cytoplasmic vs nuclear) could affect disease-specific survival of sarcoma patients. STAT1 protein expression levels were examined in 123 soft tissue sarcoma samples from human patients by immunohistochemical staining. We observed a positive correlation between the nuclear STAT1 (pSTAT1) protein level and disease-specific survival. A statistically significant inverse correlation was also observed between the cytoplasmic STAT1 protein level and disease-specific survival in sarcoma patients. We have shown that STAT1 is negatively regulated by an interferon-stimulated gene, Interferon Regulatory Factor 8 (IRF8). Disruption of IRF8 function by ectopic expression of an IRF8 dominant negative mutant resulted in dramatic up-regulation of STAT1, down-regulation of Janus kinase 1 (JAK1) and pSTAT1, and increased metastatic potential of sarcoma cells in an experimental metastasis mouse model. In addition, using microRNA microarray analysis we have identified a possible mechanism for these changes through mi106b. The possibility of cytoplasmic STAT1 and nuclear STAT1 accumulation being responsible for the observed difference in disease-specific survival of sarcoma patients merits further study of the role STAT1's localization plays in cancer progression. Direct evidence of cytoplasmic STAT1 increasing tumor cell survival, as well as confirmation of our proposed mechanism for STAT1 regulation through IRF8 is still needed. Taken together, our research suggests that IRF8 is a negative regulator of cytoplasmic STAT1 and STAT1 is indeed a tumor promoter.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2123. doi:10.1158/1538-7445.AM2011-2123
Abstract 2126: Androgen receptor regulates lung cancer progress through modulation of OCT-4 expression
Authors: Lu Hsuan-Hsuan , Yeh Shauh-Der , Chou Yu-Ting , Tsai Yu-Ting , Chang Chawnshang , Wu Cheng-Wen
Published: July 2012

Lung cancer is the leading cause of cancer mortality worldwide with unclear pathogenesis. The annual age-adjusted cancer incidence rates of lung cancer in male is higher than female in the U.S. But the role of androgen/AR in lung cancer has not been demonstrated. Immunohistochemical stain and RT Q-PCR studies demonstrated the overexpression pattern of AR in patients with non-small cell lung cancer (NSCLC) especially in male. Then we focused on the role of AR in lung cancer progression. Knockdown AR by lentivirus infection in NSCLC cell lines significantly suppressed cell proliferation and anchorage-independent growth in soft agar assay through the down-regulation of cyclin D1 expression. Besides, knockdown AR could also suppress the migration and invasion ability in CL1-5 and H1299 cells. In immunodeficient mice, AR-knockdown NSCLC cells showed dramatically decreased tumorigenesis and metastasis ability compared with control cells. Interestingly, we also found that the down-regulation of OCT-4 protein expression in AR knockdown CL1-5 and H1299 lung cancer cells. In conclusion, our data suggest that AR may play important roles in tumor growth and metastasis through its interaction with stemness pathway and it also implies AR pathway may become one of novel therapeutic targets in NSCLC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2126. doi:10.1158/1538-7445.AM2011-2126