bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 473: Elevated cAMP decreases migratory potential in pancreatic cancer cells
Authors: Zimmerman Noah , Roy Ishan , Wilson Jessica , Dwinell Michael
Published: August 2012

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, primarily resulting from inherent, aggressive dissemination. As elevated concentrations of cAMP are known to inhibit cell movement in multiple tissues, we investigated the role of cAMP in regulating migration of model pancreatic cancer cells. Increased cAMP concentrations were induced either through forskolin, an activator of adenylyl cyclase, the cAMP analog 6-bnz-cAMP, or by two phosphodiesterase cyclic nucleotide inhibitors, cilostamide or IBMX. Transwell migration assays demonstrated cAMP elevating agents significantly decreased migration of Panc1, MiaPaCa2, and HPAFII pancreatic cancer cell lines. In Panc1 cells, inhibition was dose-dependent and not significantly different between forskolin, cilostamide, or IBMX treatment. Importantly, a significant decrease in cell migration was also observed in cells treated with 6-bnz-cAMP, a specific activator of protein kinase A. The decrease in migration did not reflect alterations in cell proliferation as cell cycle analysis demonstrated little to no change in proliferation in treated compared to control cells. Similarly, the decrease in cell migration was not a function of programmed cell death as apoptosis was not elevated in cells treated with cAMP elevating agents. Consistent with its known role in F-actin rearrangement, elevated levels of cAMP increased phosphorylated RhoA and the resultant decrease in active RhoA-GTP. As expected, fluorescence microscopy indicated a significant increase in cortical actin formation along the perimeter of forskolin or cilostamide-treated cells. Taken together these data suggest that the novel use of existing anti-inflammatory drugs that target the cAMP signaling networks have the potential to block pancreatic cancer dissemination.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 473. doi:1538-7445.AM2012-473
Abstract 4732: Repression of transcriptional activity by the retinoid target gene G0S2
Authors: Ma Tian , Dong Jessica P. , Blumen Steven , Fei Dennis L. , Henderson Michael , Sekula David J. , Freemantle Sarah J. , Dmitrovsky Ethan
Published: August 2012

Acute promyelocytic leukemia (APL) cases that express the PML/RARα fusion protein block maturation of myeloid precursors. All-trans retinoic acid (RA)-based differentiation therapy of these APL cases causes clinical remissions. Yet, clinical use of RA is associated with toxicities and drug resistance. In the search for RA target genes that can serve as alternative therapeutic targets in APL, G0S2 was found as one of the most rapidly and prominently RA-induced species. G0S2 is a 103 amino acid protein without apparent homology domains to other species. It was recently shown to inhibit adipose triglyceride lipase (ATGL) activity, leading to repression of lipolysis. G0S2 is abundantly expressed in adipose tissues, which is consistent with its role in regulating lipolysis. It is also induced in response to inflammatory stimuli in other cell types, indicating it has additional roles beyond fat storage. Here, we report a new G0S2 function uncovered after its transient transfection that led to substantial repression of reporter activities of multiple constructs (including RA-regulated species RARα, UBE1L and G0S2 itself). This repression was antagonized by siRNA-mediated G0S2 knockdown. Notably, an engineered form of G0S2 that lacked the internal hydrophobic domain did not exhibit these repressive effects. Inhibitors of either the proteasomal or lysosomal pathways did not overcome this suppression by G0S2. Thus, these repressive activities were unlikely due to enhanced proteasome or lysosome activities. The reported inhibition of ATGL activity by G0S2 causes an increase in lipid droplet size, which can also be achieved by treating cells with the neutral lipid oleic acid. Yet, this treatment did not impact reporter activity, indicating that changes in lipid droplet size did not affect this transcriptional repression. Intriguingly, G0S2 expression did not reduce endogenous expression of RARα, UBE1L or G0S2 (each RA-regulated species), indicating that only exogenous genes were affected by G0S2 transient transfection. Subcellular localization of endogenous G0S2 was examined in RA-treated NB4 APL cells and G0S2 was markedly expressed in the cytosol and membranes of these cells. Transiently transfected G0S2 protein was also detected in the cytoplasm. In summary, we report a previously unrecognized function for the RA target gene G0S2: it repressed exogenous reporter activity, but not endogenous gene expression. Both endogenous and exogenous G0S2 proteins were detected in the cytoplasm. Mechanistic studies that engage this G0S2 repressive effect are now underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4732. doi:1538-7445.AM2012-4732
Abstract 4737: Resveratrol targeting of carcinogen-induced brain endothelial cell inflammation biomarkers MMP-9 and COX-2 is Sirt1-independent
Authors: Annabi Borhane , Lord-Dufour Simon , Vezina Amelie , Lamy Sylvie , Beliveau Richard
Published: August 2012

Background: The occurrence of a functional relationship between the release of metalloproteinases (MMPs) and the expression of cyclooxygenase (COX)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB), increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Methods: Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC) were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Results: Tissue scan microarray analysis of grade I-IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I-III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA), a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IαB phosphorylation in siSirt1-transfected HBMEC was however observed. Conclusion: Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4737. doi:1538-7445.AM2012-4737
Abstract 4742: Comprehensive epitope mapping of antibodies against CXCR4 and Claudin-4 by Shotgun Mutagenesis
Authors: Paes Cheryl , Rucker Joseph , Thaker Yana , Cameron Alex , Maksimchuk Ken , Payne Riley , Doranz Benjamin J.
Published: August 2012

Identifying antibody epitopes on membrane proteins can help differentiate antibody binding mechanisms, identify immunodominant structures, distinguish and classify antibodies, and identify cancer state-specific antigens, providing valuable information for therapeutic and diagnostic applications. However, membrane proteins represent challenging targets for epitope mapping studies using conventional methodologies such as X-ray crystallography or peptide scanning. We have developed a technology, ‘Shotgun Mutagenesis Epitope Mapping’ for rapidly mapping antibody epitopes on structurally-intact proteins expressed in mammalian cells. Here, we applied Shotgun Mutagenesis to create a comprehensive mutation library for CXCR4, a GPCR associated with tumor metastasis. The CXCR4 mutation library, which comprised >700 clones with 2.7x coverage of each of the 352 residues, was screened in triplicate with 5 different anti-CXCR4 MAbs to identify their epitopes. Our analysis revealed that all CXCR4 MAb epitopes were conformational and showed a requirement for charged residues located on the second extracellular loop. CXCR4 MAb epitopes were mapped onto the crystal structure to visualize and identify common structural features. Using epitope information, we generated point mutations in CXCR4 at positions of critical contact residues, incorporated them into virus-like particles (‘Lipoparticles’) and measured their binding kinetics. These approaches helped define the electrostatic contribution of individual amino acid residues on CXCR4 to the association rate, dissociation rate, and overall binding interaction with CXCR4 MAbs. Taking the same approach, we generated a comprehensive mutation library for the biomarker Claudin-4, a tight junction protein upregulated in epithelial cancers. The Claudin-4 mutation library, which comprised >400 clones with complete coverage of each of the 209 residues, was screened with a panel of 7 different anti-Claudin-4 antibodies to map their epitopes. We identified several distinct conformational epitopes that all map exclusively to the large first extracellular loop, identifying this domain as an immunodominant region of claudin-4.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4742. doi:1538-7445.AM2012-4742
Abstract 4743: Exosomes in motion: Visualizing cancer metastasis via the Synaptotagmin C2B domain
Authors: Saludes Jonel , Morton Leslie , Ghosh Nilanjan , Beninson Lida , Chapman Edwin , Fleshner Monika , Yin Hang
Published: August 2012

Exosomes play pivotal roles in intercellular communication and cancer progression. Recent findings have shown exosomes to incite proangiogenesis and prepare lymph nodes as remote niches for accumulation and migration of melanoma cells. Exosomes present a highly curved morphology with a size range of ∼30-100 nm that is distinct from other lipid vesicles. Furthermore, exosomes have the potential as a general metastasis biomarker since their increased secretion in the peripheral blood has been correlated with cancer metastasis. Since aqueous suspensions of exosomes cannot be seen by conventional optical methods, we hypothesized that a molecular probe that binds to exosomes could be a novel tool for detecting these vesicles. We aim to develop a minimally-invasive diagnostic tool to detect and measure secreted exosomes as biomarkers of cancer metastasis that may help clinicians and patients decide on an appropriate therapeutic action. Taking a cue from nature and using solid phase ‘Click’ chemistry as a technique for peptide modification, we created a fluorophore-tagged cyclic peptide based on the membrane penetrating C2B domain of Synaptotagmin I. We investigated the peptide-lipid interactions using synthetic liposomes as preliminary models of exosomes through a combination of spectroscopic techniques. We found that the peptide probe was selective for highly curved liposomes (d <100 nm) and does not bind to low curvature liposomes (d >100 nm). We tested blood plasma samples from rat models to find out if our preliminary findings would translate to the detection of exosomes. Our results showed that blood plasma treated with the peptide probe showed fluorescence intensity that was remarkably higher than the untreated peptide, which indicated binding to exosomes. Furthermore, we used a real time nanoparticle tracking analysis system to visualize the exosomes in a dynamic and physiologically-relevant medium. The peptide-treated exosomes were detected as fast-moving, fluorescent particles even in the complex matrix of blood plasma. This technique provided us with a platform to study the size distribution and concentration of exosomes in blood plasma. We have created a peptide probe that selectively binds to exosomes from an animal model. The investigation of the exosome binding property of this probe on clinically relevant samples is ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4743. doi:1538-7445.AM2012-4743
Abstract 4746: Structural studies of human glioma pathogenesis-related protein 1(GLIPR1)
Authors: Asojo Oluwatoyin A. , Koski Raymond A. , Bonafé Nathalie
Published: August 2012

Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide, to direct its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory protein, antigen 5, pathogenesis related-1) domain, and a transmembrane domain. Phase I/II clinical trial involving direct injection of neoadjuvant GLIPR1 prior to radical prostatectomy are underway (IND #13033). GLIPR1 gene therapy has been shown to be anti-metastatic in an orthotopic mouse model of prostate cancer. GLIPR1 is also currently being investigated for glioblastoma targeted therapy. We present here crystal structures of a truncated soluble domain of the human GLIPR1 protein, sGLIPR1, solved by molecular replacement (MR) using a truncated polyalanine model of the CAP domain of stecrisp, a snake venom CRISP (cysteine-rich secretory protein), in PHASER. The correct MR solution could only be obtained by removing all loops in the search model. While, the native structure was refined to 1.85 Å, that of the Zn2+ complex was refined to 2.2 Å. The latter structure reveals that the putative binding cavity coordinates Zn2+ similarly to snake venom CRISPs which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop / turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. We also propose a model for the structure of the full-length, membrane-bound GLIPR1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4746. doi:1538-7445.AM2012-4746
Abstract 4748: Molecular targets of quinolinone derivatives with anticancer activity
Authors: Rylova Gabriela , Dzubak Petr , Janostakova Anna , Frydrych Ivo , Konecny Petr , Holub Dusan , Ozdian Tomas , Dolezal Dalibor , Soural Miroslav , Hlavac Jan , Hajduch Marian
Published: August 2012

Currently, we are testing chemical libraries of quinolinone derivatives, compounds with high selective cytotoxic activity. These compounds, derived from quinolone antibiotics, are well known for their antibacterial, antiprotozoal, cytotoxic and immunosuppressive effects. 2-phenyl-3-hydroxy-4(1H)-quinolinones were tested by MTT assay for cytotoxic activity and some of them proved IC50 values in sub-micromolar concentrations. Cell cycle and DNA/RNA synthesis were further analysed by flow cytometry. The accumulation of cells was observed predominantly in G1 phase together with significant DNA and RNA synthesis inhibition. The most active derivative was tested in in vivo cancer mice model with using hollow fibres. Hollow fibres were filled by leukemic CEM cell line and were implanted subcutaneously and intraperitoneally to mice. Afterwards, mice were treated by tested compound. Hollow fibres were after 14 days taken from mice and surviving cells were tested by cytotoxic MTT assay. activity of tested quinolinone was comparable to doxorubicin. For protein targets identification, we have synthesized biotinylated molecules of original derivatives. Biotinylated derivatives were immobilized on the surface of streptavidin coated magnetic beads and whole complex (magnetic bead-streptavidin-biotin-quinolinone) was incubated with total cellular lysate of the CEM cell line. Thereafter non-bonded proteins were washed-out and specifically bonded proteins were separated on SDS-PAGE gel. Following silver-staining of the gel we identified sample lines with specifically bonded proteins to biotinylated compound and negative control. Specific proteins were cut off from the gel and trypsinized to get peptides for protein identification by MS analysis (LC-MS/MS and LC-MALDI). Identified targets are proteins associated with translational regulation, glucose metabolism and cytoskeleton. Potential targets were validated by western blot and by other assays (reporter cell models, kinase and enzymatic assays, evaluation in vitro transcription and translation). The work was supported by grants: LC07017, CZ.1.07/2.3.00/20.0009 coming from European Social Fund. Infrastructural part of this project (Institute of Molecular and Translational Medicine) was supported from the Operational Programme Research and Development for Innovations (project CZ.1.05/2.1.00/01.0030).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4748. doi:1538-7445.AM2012-4748
Abstract 4755: The role of a pair of cysteine residues in the redox properties of human ribonucleotide reductase small subunits hp53R2 and hRRM2
Authors: Zhou Bingsen , Su Leila , Hu Shuya , Yen Yun
Published: August 2012

Ribonucleotide reductase (RNR) is a cell cycle protein regulating intracellular redox state, which is a critical mediator and metabolic modulator of cellular functions such as cell signaling, gene expression, proliferation, and apoptosis. Human RNR has two small subunits hp53R2 and hRRM2, which by themselves play important roles in regulating cellular redox state. Our previous studies have shown that hRRM2 and hp53R2 have different redox properties, where hp53R2 is an antioxidant defending oxidative stress while hRRM2 tends to be an oxidant. Many proteins consist of cysteine residues which have a sulfhydryl group with a labile proton, making it an ideal “receptor” through the reversibility of the thiol/disulfide redox couple. In this work we have found human RNR small subunits possess a conserved CXXXC motif, C270XXXC274 in hRRM2 and C232XXXC236 in hp53R2, close to Tyr162-hRRM2/Tyr124-hp53R2. Our mutagenesis study indicated this motif is linked with the redox properties of hp53R2 and hRRM2. The cysteines were mutated to serine and alanine respectively and the redox state of the mutants were monitored by the fluorescence assays of carboxy-H2DCFDA oxidation. Electron paramagnetic resonance spectroscopy (EPR) and enzyme activity assays were also carried out for the radical stability and enzyme activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4755. doi:1538-7445.AM2012-4755
Abstract 4756: YM155 suppresses survivin expression by disrupting the ILF3/p54nrb transcription factor complex
Authors: Nakamura Naoto , Yamauchi Tomohiro , Hiramoto Masashi , Yuri Masatoshi , Naito Masanori , Takeuchi Masahiro , Yamanaka Kentaro , Nakahara Takahito , Kita Aya , Kinoyama Isao , Matsuhisa Akira , Sasamata Masao , Yokota Hiroyuki , Kawabata Shigeki , Furuichi Kiyoshi
Published: August 2012

Survivin, a member of the anti-apoptosis proteins family, is highly expressed in all primary tumor types, and is responsible for cancer progression and drug resistance in many types of cancer. YM155, a small chemical compound, selectively suppressed the expression of survivin and induced apoptosis in cancer cells both in vitro and in vivo. However, the mechanisms underlying the suppression of survivin expression by YM155 are unknown. In order to identify the molecular targets and analyze the drug mechanism of action, affinity purification was performed using an active analogue of YM155. We identified interleukin enhancer-binding factor 3 (ILF3) as the binding target of YM155. From the complex analysis of ILF3 and survivin promoter sequence, we also found that ILF3 forms a complex with p54nrb transcription factor, and then binds to the survivin promoter. Overexpression of ILF3 enhanced survivin promoter activity, which was attenuated by YM155 in a concentration-dependent manner. Furthermore, the ILF3/p54nrb complex was disrupted by YM155, thus dispersing the components in the nucleus.In conclusion, our study suggests that binding to ILF3 by YM155 causes the dissociation of the ILF3/p54nrb complex, thus inhibits ILF3 dependent survivin expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4756. doi:1538-7445.AM2012-4756
Abstract 4757: Design and optimization of linked nucleic acid (LNA) probes to detect the c-MYC G-quadruplex
Authors: Gaerig Vanessa C. , Jin Xi , Tabrizian Tahmineh , Danford Forest L. , Brooks Tracy A.
Published: August 2012

The c-MYC oncogene is upregulated in almost 80% of cancers, and in some cases is the causative oncogenic factor. It is regulated at the transcriptional level with up to 90% initiating at the P1 and P2 promoters. The NHE III1 element regulates this region, which consists of a unique guanine-rich string of DNA capable of adopting at least three topologies: single stranded, double stranded (ds) and the non-B-DNA G-quadruplex (G4) structure. G4s are formed when two or more tetrads stack, each comprised of four guanines bonded by Hoogsteen hydrogen bonds, stabilized with monovalent cations. Putative G4 forming regions have at least four runs of three or more consecutive guanines separated by varying nucleotides that comprise the loop structures. The c-MYC G4 structure has been elucidated to involve guanine runs 1α4 (G41-4) or 2Δ5 (G42-5) (of six total runs) in a parallel formation. Regulatory proteins have been identified, and small molecule compounds are under investigation to stabilize the G4 element and downregulate c-MYC expression. In parallel to these efforts, we are developing linked nucleic acid (LNA)-biotin bridge probes to be used as diagnostic tools to identify these formations in biopsies in order to inform therapeutic regimens. Molecular models of the two G4 isoforms were used to determine the distance occupied by the secondary structure to be between 16.4α16.6 angstroms; various length nucleic acid sequences flanking a 17.7α30 angstrom linker were screened and optimized. Probes were shown to be structure specific - either for the c-MYC G41-4 (probe 1a) or the G42-5 (probe 1b) - by EMSA analysis. The probes were amenable to internal biotinylation for ongoing pull-down assays, but truncation of the 5′-complementary sequence limited affinity. Interestingly, the LNA probes thermally stabilized the c-MYC G4 in a unique pattern and are also being examined for their potential therapeutic efficacy. Experiments are underway to show that these hybrid probes will bind intracellularly and show the frequency of G4 formation in quiescent versus activated cells, and following treatment with c-MYC G4-interactive compounds. Through these works, we highlight for the first time that a ‘FISH’-type probe can be developed and used to selectively detect the formation of the c-MYC G4 in vitro. Up to 80% cancer patients overexpress c-MYC and are candidates for G4-targeted therapies currently under development. The developed probes will be of great utility as companion diagnostics in selecting patients for G4-therapeutics and truly personalized medicine.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4757. doi:1538-7445.AM2012-4757
Abstract 4767: Reaction of singlet oxygen with hydrazone prodrugs for the photochemical release of N-nitrosoamine drugs
Authors: Ghogare Ashwini A.
Published: August 2012

Very few visible-light activated photocleavable groups are available for drug delivery applications. Our research was interested in assessing the visible-light photolability of hydrazone C=N bonds via a Type II (singlet oxygen) dye-sensitized oxygenation reaction. We have explored the photooxidized cleavage of N-nitrosoamine by-products from N-methylbenzophenone phenylhydrazone and methylenehydrazine model compounds. The former was studied by gas chromatography mass spectrometry and the latter by density functional theory calculations (DFT). We quantified benzophenone that arose from the phenylhydrazone photooxidation. The DFT calculations predicted a low energy route to C=N bond fragmentation via a [2+2] pathway and an 1,2,3-dioxazetidine reactive intermediate. The reaction was predicted to occur in stepwise process via zwitterionic intermediates prior to forming the dioxazetidine, and cleavage to the nitrosamine product. Further studies are underway regarding the photolytic and hydrolytic stability of the hydrazone C=N bond to give better insight including structure-activity relationships, of singlet oxygen triggered photo-release of nitrosoamines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4767. doi:1538-7445.AM2012-4767
Abstract 4774: LC-MS/MS detection of the dansyl-modified biologically active CDK inhibitor VMY-1-103 using an in vivo model system
Authors: Sirajuddin Paul , Das Sudeep , Ringer Lymor , Salinas Patricia , Rodriguez Olga , Moasser Bahram , Yenugonda Venkata M. , Albanese Christopher , Brown Milton
Published: August 2012

The development of new small molecule kinase inhibitors as cancer therapeutic drugs has placed increased emphasis on determining the mechanism of action of the new compounds preclinically, such as in genetically engineered mice or using human tissue xenografts. The ability to accurately measure the pharmacokinetics and pharmacodynamics of new compounds is often hampered by the lack of suitable platforms to measure serum and tissue distribution with high sensitivity and specificity. We have recently developed a novel dansylated cyclin dependent kinase inhibitor called VMY-1-103 (VMY) that has purvalanol B as its base compound. We established that VMY was significantly more effective in blocking cell cycle progression and inducing apoptosis than purvalanol B. Since dansylation is frequently used to enhance mass spectrometry-based quantitative analyses, we hypothesized that this modality could similarly be used to evaluate the tissue distribution of VMY in mouse models. In the current study, we developed a rapid liquid phase-extraction of VMY and purvalanol B from mouse tissue and validated a sensitive liquid chromatography-tandem mass spectrometry based assay to quantify tissue delivery of these compounds in vivo. Comparisons of the standard curves for VMY versus Purvalanol B revealed a greater than 7.7-fold increase in sensitivity for VMY. Using both the ND-2 SmoA1, a genetically engineered mouse model of medulloblastoma, and normal mice, we quantified tissue delivery by mass spectrometry as well as Area Under the Curve (AUC) analysis by integrating the peak intensity normalized by an internal standard. Our analyses revealed that VMY delivered intraperitoneally at 20mg/kg was found at its highest concentration in serum 4 hours after injection. We further established that VMY was distributed in multiple tissues, including pancreas and prostate at varying levels and, importantly, we establish that VMY was able to traverse an intact blood-brain barrier as it was detected in the brain and cerebellum of normal mice. Purvalanol B delivered in the same manner as VMY was found at significantly lower levels in all tissues examined and unlike VMY, we failed to detect purvalanol B in brain or and cerebellar tissue. The significance of this study illustrates that a biologically active compound can be modified by dansylation and exhibit both increased sensitivity to mass spectrometry and enhanced bioavailability and tissue delivery, including the brain, in vivo. We also define a simple mass spectrometry-based tissue and serum assay.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4774. doi:1538-7445.AM2012-4774
Abstract 4777: Computational strategies for finding protein targets of flavonoids
Authors: Chen Hanyong , Yao Ke , Nadas Janos , Bode Ann M. , Malakhova Margarita , Oi Naomi , Lubet Ronald A. , Dong Zigang
Published: August 2012

Plant-based polyphenols (i.e., phytochemicals) have been used as treatments for human ailments for centuries. The mechanisms of action of these plant-derived medicines are a now major topic of investigation. Thousands of phytochemicals have been isolated, and a certain number of them have shown activities or effects on different disease models. Using conventional approaches to select the best or group of the best chemicals for studying the effectiveness in treating or preventing disease is extremely challenging. We have developed and used computational-based methodologies that provide an efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by polyphenols. Computational strategies involving virtual screening, shape and pharmacophore analysis and molecular docking have been used to select chemicals that target a particular protein or enzyme and to determine potential protein targets for well-characterized as well as for novel polyphenols.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4777. doi:1538-7445.AM2012-4777
Abstract 478: Does ICAM-2 contribute to the metastatic phenotype of pancreatic cancer cells
Authors: Garcia Patrick L. , Yoon Karina J.
Published: August 2012

Pancreatic Cancer (PC) is the 10th most diagnosed cancer and the 4th leading cause of cancer related deaths in the United States, and of those deaths, approximately 90% are due to metastatic disease. Metastasis involves many different proteins and pathways, but one family of proteins, cell adhesion molecules (CAMs) have been implicated in the metastatic process. CAMs are membrane bound glycoproteins involved in cell adhesion to other cells or the extracellular matrix. A specific protein within the CAM superfamily is intercellular adhesion molecule 2 (ICAM-2), which was shown to be expressed in leukocytes and vascular endothelium. Previously, our lab reported a novel function for ICAM-2 in neuroblastoma (NB) cells. ICAM-2 expression correlated with decreased cell motility in vitro, a decreased capacity to form disseminated tumors in vivo in an experimental metastasis model, and coincided with a favorable prognosis in primary tumor samples. The objective of our current study is to examine if these findings could be expanded to other types of solid tumors. Interestingly, in a preliminary screen for ICAM-2 expression, we detected ICAM-2 expression in other types of solid tumors, including PC cell lines. We detected RNA and protein expression of ICAM-2 in two cell lines (BxPC-3 and CFPAC-1), while ICAM-2 expression was not detectable in Panc-1 cells. Next, we wanted to determine if the absence of ICAM-2 expression correlated with increased colony formation in anchorage independent growth in soft agar assay, which was previously reported to be an in vitro indicator of either tumorigenic or metastatic potential. Surprisingly, ICAM-2 expressing PC cells showed significantly (p<0.001) decreased colony growth compared to non-ICAM-2 expressing cells. Currently, we are i) evaluating functional differences between ICAM-2 expressing and non-ICAM-2 expressing PC cell lines, and ii) producing a stable transfected Panc-1 cell line over-expressing ICAM-2 to further assess its potential role in metastasis. This work was supported by UAB - comprehensive cancer center - young supporters board - new faculty development award 2010.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 478. doi:1538-7445.AM2012-478
Abstract 4785: Comparative proteomic analysis of MCF7 and SKBR3 breast cancer cell lines
Authors: Mojtahedi Zahra , Erfani Nasrollah , Ghaderi Abbas
Published: August 2012

Differentially expressed proteins among cancer cell lines fit each cell line as a model for gaining knowledge of heterogeneity in cancer. The aim of our study was to identify differentially expressed proteins between MCF7 (ER+, low HER2 expression) and SKBR3 (ER-, high HER2 expression) cell lines by a proteomic approach. Moreover, a number of proteins in MCF7 cell line were randomly selected and identified. MCF7 and SKBR3 cell lysates were subjected to two dimensional gel electrophoresis and spots of interest were identified by MALDI-TOF/TOF mass spectrometry. Upregulated proteins (≥2 fold and p value <0.05) in MCF7 cells were cellular retinoic acid binding-protein 2, Hsp27, nucleophosmin, electron transfer flavoprotein ≤ subunit, and profilin, and in SKBR3 cells were Rho GDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel 2, aldehyde dehydrogeanase-2 (ALDH2), LDH- A, LDH-B, pyrophosphates 1, GAPDH, cathepsin D preprotein, and apolipoprotein A-I binding. Most of the identified proteins have been a candidate marker for cancer aggressiveness or drug resistance, but their differential expressions between SKBr3 and MCF7 cells were not known. Apo-lipoprotein binding-protein has not been described in cancer so far. Differential expression of RhoGDI-α was further validated by using western blotting with specific antibody. Further studies are required to clarify the importance of differential expressions of the identified proteins in SKBr3 and MCF7 breast cancer cell line models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4785. doi:1538-7445.AM2012-4785
Abstract 479: The effects of sarcophine-diol (SD) on melanoma B16F10 cell line
Authors: Szymanski Pawel T. , Kuppast Bhimanna , Muley Pratik , Fahmy Hesham
Published: August 2012

Sarcophine diol (SD) is a non-toxic semi-synthetic derivative of sarcophine, a marine natural product. Previously, we studied the chemopreventive effects of SD on non-melanoma tumor development using both animal and cellular models. In this current study, we used the mouse melanoma B16F10 cell line to investigate the possible effects of SD on melanoma cell development. We found that SD at a 250 µM concentrations inhibits cell duplication. Melanoma cells treated with SD do not form the contractile ring, a multi-protein complex involved in cell division. As a result of cytoskeleton modification, cells treated with SD do not show the ability to carry out de novo synthesis of DNA, and their DNA show a high degree of fragmentation compared to untreated controls. To explore the molecular mechanism(s) involved in the disassembly of the contractile machinery, our studies show that SD treatment inhibits cell membrane permeability for small molecular mass compounds (e.g. ethidium bromide which is used as an indicator for cell permeability to Ca2+ ions). These findings are consistent with the observation that SD increases cleavage of phospholipase A2 (PLA2), resulting in low PLA2 enzymatic activity. These findings are also consistent with the observation that SD, in a time- and concentration-dependent manner, inhibits the expression level of the cyclooxygenase-2 (Cox-2). As a result of the decline in membrane permeability in SD treated cells and the negative effect of SD on the protein level of phospholipase C (PLC), which both affect the activity of contractile machinery, the non-dividing cells undergo apoptosis. The latter notion is supported by findings that SD inhibits the expression of STAT-3 (signal transducer and activator of transcription protein), and cyclin D1 (an activator of cyclin-dependent kinase 4, Cdk4). SD treatment also enhances the cellular level of the tumor suppressing protein 53 (p53) and stimulates cleavage of the nuclear poly(ADP-ribose) polymerase (cleaved-PARP). SD also enhances the cellular level of cleaved-Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. All these findings, in addition to inhibition of cell viability, suggest that SD most likely inhibits melanoma cell growth by arresting the cell-division cycle in a Go quiescent phase and simultaneously activates apoptosis via extrinsic and intrinsic pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 479. doi:1538-7445.AM2012-479
Abstract 4795: Metabolic biomarkers of hepatocellular carcinoma
Authors: Ressom Habtom W. , Xiao Jun Feng , Tuli Leepika , Varghese Rency S. , Zhou Bin , Tsai Tsung-Heng , Ranjbar Mohammad R. Nezami , Wang Jinlian , Cheema Amrita K. , Tadesse Mahlet G. , Goldman Radoslav , Shetty Kirti
Published: August 2012

Characterizing the metabolic changes pertaining to hepatocellular carcinoma (HCC) in patients with liver cirrhosis (LC) is believed to contribute towards early detection, treatment, and our understanding of the molecular mechanisms of HCC. Prospectively evaluated adult patients with LC were recruited from our hepatology practice. All patients were diagnosed to have LC on the basis of established clinical, laboratory and imaging criteria. Cases were diagnosed to have HCC based on either histology or characteristic imaging features. The LC controls were required to be HCC free for at least 6 months from the time of study entry. We compared metabolite levels in sera of 78 HCC cases with 184 LC controls by using ultra performance liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from LC controls were selected by statistical methods. Putative metabolite identifications for these ions were obtained through mass-based database search. Pathway analysis of these putative identifications reveals significant enrichment of metabolites involved in bile acid biosynthesis, porphyrin and chlorophyll metabolism, and arachidonic acid metabolism. Verification of the identities of selected metabolites was conducted by comparing their MS/MS fragmentation patterns and retention time with those from authentic compounds. Quantitation of these metabolites was performed in a subset of the serum samples (10 HCC and 10 LC) using isotope dilution by selected reaction monitoring (SRM) on triple quadrupole linear ion trap (QqQLIT) mass spectrometer. The results of this analysis confirms that metabolites involved in phospholipid catabolism such as sphingosine-1-phosphate (S-1-P) and lysophosphatidylcholine are up-regulated in sera of HCC vs. those with LC. Down-regulated metabolites include those involved in cholesterol metabolism such as glycochenodeoxycholic acid 3-sulfate (3-sulfo-GCDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), taurochenodeoxycholate (TCDCA), as well as those involved in heme catabolism including billuribin, I-Urobili, and bilirubin glucuronide. The preliminary results obtained in this study provide useful insights into our understanding of metabolic differences between HCC and LC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4795. doi:1538-7445.AM2012-4795
Abstract 4861: Triple-negative breast cancers establish a systemic environment that drives malignant progression of otherwise indolent disseminated tumors via EGF and IGF
Authors: Castano Zafira , Marsh Timothy , Tadipatri Ramya , Kuznetsov Hanna , Al-Sahour Fatima , Paktinaz Mahnaz , McAllister Sandra S.
Published: August 2012

Breast cancer recurrence rates are variable, suggesting that tumor cells disseminate from primary sites at an early stage but remain indolent for extended periods of time before progressing to symptomatic disease. Little is known about mechanisms that cause otherwise dormant tumors to become overt cancers, making it difficult to predict which breast cancer patients are likely to relapse and therefore likely to benefit from preemptive therapy. To address these challenges, we use a human breast tumor xenograft model in which aggressively growing breast cancers (“instigators”) promote the growth of otherwise indolent breast cancers (“responders”) that have disseminated to distant sites. These pro-tumorigenic systemic effects are established by instigating tumors, in large part, through their mobilization of bone marrow cells (BMCs) that are subsequently recruited to the responding breast tumors, where they create a tumor-supportive microenvironment. We report here that the mechanisms of establishing tumor-supportive systemic environments depend upon the molecular-histopathological subtype of the instigating breast cancer. Instigating cell lines and primary tumor surgical specimens, derived from patients with either triple-negative breast cancer (TNBC) or luminal breast cancer (LBC), established different subtype-specific systemic environments that impinged in different ways upon the malignant features of responding tumor cells and their microenvironment. In particular, the TNBC-induced responding tumor microenvironment was characterized by elevated levels of epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF1). EGF and IGF acted on responding tumor cells in a paracrine manner to induce expression of factors that play a role in tumor-initiation, embryonic stem cell maintenance, and epithelial-mesenchymal transition. Pharmacological administration of EGFR and IGF1R inhibitors prevented responding tumors from acquiring the TNBC-induced malignant features, thus maintaining them in an indolent state. Our results demonstrate that microenvironmental composition, as well as tumor cell phenotypic plasticity and molecular properties, are strongly influenced by the host systemic macroenvironment. Our work suggests that the crosstalk between disseminated tumor cells and their microenvironment can be exploited therapeutically to improve outcomes for patients with TNBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4861. doi:1538-7445.AM2012-4861
Abstract 4888: Decitabine and vorinostat cooperate to sensitize metastatic human colon carcinoma cells to Fas-mediated apoptosis
Authors: Torres Christina M. , Yang Dafeng , Zimmerman Mary , Bardhan Kankana , Liu Kebin
Published: August 2012

The death receptor Fas and its physiological ligand, FasL, which is expressed on the surface of cytotoxic T-cells (CTLs), regulate apoptosis of cancerous cells. While normal colon epithelial cells express high levels of Fas, Fas expression is diminished in colon carcinomas. Metastatic colon cancers acquire an apopotosis-resistant phenotype often due to the loss of Fas expression and function. Previous studies have implicated Fas-mediated apoptosis as an important contributor of tumor regression. Therefore, targeting Fas resistance is of critical importance in treating colon cancer through Fas-based cancer therapy and immunotherapy. In this study, we explore whether epigenetic pharmacological agents can restore Fas expression and re-establish sensitivity to Fas-mediated apoptosis in metastatic human colon carcinoma cells. First we determined that the epigenetic drugs Decitabine, a DNA methyltransferase inhibitor, and Vorinostat, a HDAC inhibitor, each are capable of up-regulating Fas expression. When combined, these two inhibitors cooperate to increase Fas expression to an even greater extent than each agent alone. Pre-treatment with decitabine, vorinostat, or both drugs followed by incubation with FasL, was able to overcome resistance to FasL-induced apoptosis. Analysis of bisulfite-modified genomic DNA, isolated from human metastatic colon carcinoma cells revealed that the Fas promoter is sporadically methylated; therefore sensitization of Fas receptor expression on the cell surface by decitabine was unlikely through direct inhibition of Fas promoter meythlation. Although the Fas promoter was not heavily methylated, the promoters for BNIP3 and Bik, which are downstream of Fas signaling, were methylated. After treatment with these pharmacological epigenetic inhibitors, alone or in combination, protein levels of both BNIP3 and Bik were increased. Our data suggest that combined modalities of chemotherapy and Fas-based immunotherapy may be an effective approach for the suppression of colon cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4888. doi:1538-7445.AM2012-4888