bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1593: Cellular effects of the mitochondrially-directed antioxidant Tempol.
Authors: Kesarwala Aparna H. , DeGraff William , Gamson Janet , Choudhuri Rajani , Cook John A. , Krishna Murali C. , Mitchell James B.
Published: August 2014

The mitochondrial respiratory chain is a major source of reactive oxygen species, making it a high-yield target for antioxidants and potential radioprotection. The lipophilic cation triphenylphosphonium (TPP) has been used to target antioxidants to the mitochondrial membrane. Its lipophilicity permits it free passage through biological membranes while its positive charge traps it in the mitochondrial membrane, which is characterized by a large membrane potential. Among the agents localized to mitochondria via TPP have been stable nitroxide radicals, superoxide dismutase mimetics which also demonstrate the ability to scavenge free radicals. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, or Tempol (TP), is a nitroxide which is radioprotective both in vitro and in vivo when present during ionizing radiation. As with most chemical radioprotectors, relatively high concentrations of Tempol are required for protection. We questioned whether mito-Tempol (MTP), Tempol directed to mitochondria via TPP, would provide protection against radiation or hydrogen peroxide (HP) cytotoxicity as measured by the clonogenic assay in Chinese hamster V79 cells. MTP (10-500 μM) present 10 min prior to, during, and 4 hr after 12 Gy provided no radioprotection. MTP exhibited no cytotoxicity alone. In contrast, both TP and MTP (500 μM), when present immediately before and during a 1 hr exposure to HP, provided significant protection against HP cytotoxicity. We next evaluated the effects of continuous exposure to TP or MTP (100 μM) on the growth rate of several human tumor cell lines (MCF7, HT29, 786-0). TP had little effect on growth of these cell lines while MTP completely inhibited cell growth. The effects of the TPP-carbon linker alone will be presented; in short-term studies it did not exhibit cytotoxicity to V79 cells. These preliminary data with mitochondrial targeting of Tempol demonstrate that MTP exhibited antioxidant properties similar to TP with regard to protection against HP; at concentrations up to 500 μM, however, MTP did not protect against radiation. The latter finding is consistent with the requirement that chemical radioprotectors be present in sufficient concentration in the nucleus to afford radioprotection. Additional studies will be presented addressing the mechanism of MTP-mediated growth inhibition in human tumor cell lines.

Citation Format: Aparna H. Kesarwala, William DeGraff, Janet Gamson, Rajani Choudhuri, John A. Cook, Murali C. Krishna, James B. Mitchell. Cellular effects of the mitochondrially-directed antioxidant Tempol. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1593. doi:10.1158/1538-7445.AM2013-1593
Abstract 1603: Synthesis and in vitro evaluation of lipophilic cation conjugated photosensitizers for targeting mitochondria.
Authors: Rajaputra Pallavi , Nkepang Gregory , Watley Ryan , You Youngjae
Published: August 2014

Mitochondria-specific photosensitizers were designed by taking advantage of the preferential localization of delocalized lipophilic cations (DLCs) in mitochondria. Three conjugates, CMP-Rh, CMP-tPP, and CMP-(tPP)2, were successfully synthesized by linking the hydroxy core modified porphyrins (CMP-OH and CMP-(OH)2) to either rhodamine B (Rh B) or one or two triphenyl phosphine (tPP) cations respectively via a saturated hydrocarbon linker. Their ability for delivering photosensitizers to mitochondria was evaluated using dual staining fluorescence microscopy and staining pattern was compared with a mitochondrial specific probe. Fluorescence imaging study suggested that CMP-Rh specifically localized in mitochondria. On the other hand, CMP-tPP and CMP-(tPP)2 showed less significant mitochondrial localization. The conjugation of CMP to Rh and tPP did greatly improve intracellular uptake and in vitro photodynamic activity compared to CMP-OH. In addition, to evaluate the efficiency of the conjugates as photosensitizers, their photophysical properties and in vitro biological activities were studied in comparison to those of CMP-OH. All conjugates were capable of generating singlet oxygen at rates comparable to CMP-OH. This improved photodynamic activity might be primarily due to an enhanced cellular uptake. Our study suggests that Rh B cationic group is better at least for CMP than tPP as a mitochondrial targeting vector.

Citation Format: Pallavi Rajaputra, Gregory Nkepang, Ryan Watley, Youngjae You. Synthesis and in vitro evaluation of lipophilic cation conjugated photosensitizers for targeting mitochondria. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1603. doi:10.1158/1538-7445.AM2013-1603
Abstract 1690: Differential autophagy activation in KRAS and EGFR mutant lung adenocarcinomas.
Authors: Rodriguez Maria L. , Pulido Ines , Soucheray Margaret , Crespo Daniel , Al-shahrour Fatima , Shimamura Takeshi , Ortega Angel , Carretero Julian
Published: August 2014

Lung cancer is the leading cause of cancer deaths in western countries, and adenocarcinomas (LADs) are the most frequent histological subtype. The aberrant activation of the kinases promotes plethora of tumorigenic processes, mainly through PI3K and MAPK oncogenic pathways leading to oncogene addiction. The activation of PI3K pathway deregulates mTOR, a master kinase for cell growth and autophagy. Autophagy can be pro- or anti- tumorigenic, however its roles in protecting tumors exposed to metabolic stress under chemotherapy are considered as a survival mechanism for the tumors leading to acquired resistance. Consequently, the inhibition of autophagy is an attractive therapy to prevent the emergence of acquired resistance. Activating mutations in EGFR and KRAS are mutually exclusive and are the most frequent oncogene activation in LAD. There are considerable differences in the control of signalling pathways including PI3K-mTOR axis. Therefore, it is hypothesized that the susceptibility to autophagy differs depending on EGFR or KRAS mutational status. To test the hypothesis, we applied nutrient-starvation conditions, the most well-known inducer of autophagy, to a panel of LAD cell lines bearing known EGFR or KRAS mutations. As a positive control, cells were treated with rapamycin, which also induced autophagy inhibiting MTOR activity. Autophagy activation was analyzed by: i) Western blot of LC3-II and p62/SQSTM1 protein levels, that measures autophagy flux and autophagic protein degradation, respectively; and ii) autophagolysosome detection and lysosomal activity by fluorescent microscopy. Our data suggest that KRAS-mutant LAD cell lines are able to activate autophagy, whereas EGFR-mutant are not, under starvation stress. Furthermore, we found a potential interaction between mutant EGFR and Beclin 1 (BECN1, a protein required for the initiation of the autophagosome) at the cell membrane proximity, using co-immunolocalization by confocal microscopy and immunoprecipitation followed by Western blot. Together, these data provide a compelling rationale to investigate anti-autophagy therapy in mutant KRAS LADs and warrant further investigation in the regulation of autophagy via a novel link between Beclin1 and mutant EGFR.

Citation Format: Maria L. Rodriguez, Ines Pulido, Margaret Soucheray, Daniel Crespo, Fatima Al-shahrour, Takeshi Shimamura, Angel Ortega, Julian Carretero. Differential autophagy activation in KRAS and EGFR mutant lung adenocarcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1690. doi:10.1158/1538-7445.AM2013-1690
Abstract 1796: Analysis of TCGA RNASeq data using SpliceSeq provides a survey of alternative splicing in cancer.
Authors: Ryan Michael C. , Weinstein John N.
Published: August 2014

The vast majority of human genes express more than one transcript isoform, and aberrant splicing patterns or changes in isoform expression ratios can lead to disease, notably cancer. Next generation sequencing platforms deliver the finest resolution yet of the transcriptome, and the wealth of sequencing data produced by TCGA provides an excellent basis for a broad study of alternative splicing in cancer. We have developed an interactive, exceptionally user-friendly tool, SpliceSeq, for investigation of alternative splicing in RNASeq data. The program is a follow-up to our SpliceCenter package (http://projects.insilico.us/SpliceCenter/SpliceOverview.jsp), which provides tools for use by the bench biologist in assessing experimental observations that may result from splice variation.

Our results from SpliceSeq for TCGA RNASeq data from several cancer types will be presented. We identify pervasive changes in splicing patterns across different cancer types by comparing tumor vs. normal and clinical subgroups (e.g. tumor stage, hyper-mutator status, and MSI designation). Interesting observations include striking splice variations in APP, the amyloid beta precursor protein, and in PKM, which is centrally involved in the balance between oxidative and glycolytic metabolism in tumors. SpliceSeq is freely available at http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview.

1. Ryan MC, Cleland J, Kim R, Wong WC, Weinstein JN. SpliceSeq: A Resource for Analysis and Visualization of RNA-Seq Data on Alternative Splicing and Its Functional Impacts. Bioinformatics, 10.1093, 2012.

2. Ryan MC, Zeeberg BR, Caplen NJ, Cleland JA, Kahn AB, Liu H, and Weinstein JN. SpliceCenter: a suite of web-based bioinformatic applications for evaluating the impact of alternative splicing on RT-PCR, RNAi, microarray, and peptide-based studies. BMC Bioinformatics 9:313, 2008.

3. Kahn AB, Ryan MC, Liu H, Zeeberg BR, Jamison DC, Weinstein JN. SpliceMiner: a high-throughput database implementation of the NCBI Evidence Viewer for microarray splice variant analysis. BMC Bioinformatics 8:752, 2006.

Supported by NCI Grant No. U24CA143883. The content is solely the responsibility of the authors and does not necessarily represent official views of NCI or NIH. Also supported in part by a gift from the Mary K. Chapman Foundation and a grant from the Michael and Susan Dell Foundation honoring Lorraine Dell.

Citation Format: Michael C. Ryan, John N. Weinstein. Analysis of TCGA RNASeq data using SpliceSeq provides a survey of alternative splicing in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1796. doi:10.1158/1538-7445.AM2013-1796
Abstract 1829: Negative regulation of lncRNA GAS5 by miR-21.
Authors: Zhang Ziqiang , Mo Yin-yuan
Published: August 2014

Protein-coding genes account for only a small part of the human genome whereas the vast majority of transcripts make up the non-coding RNAs including microRNAs and long non-coding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. As master gene regulators, microRNAs can target a large number of protein-coding genes. However, it remains to be determined whether microRNAs can also target lncRNAs. In the present study, we determined whether miR-21 impacts lncRNA expression. Using lncRNA RT-PCR array carrying 83 human disease related lncRNAs, we showed that miR-21 was capable of repressing the growth arrest specific 5 (GAS5) lncRNA which was previously shown to be involved in growth arrest. This negative regulation between miR-21 and GAS5 was confirmed in breast tumor specimens. Of interest, GAS5 can also repress miR-21 expression. While ectopic expression of GAS5 suppressed, GAS5 siRNAs increased miR-21 expression. Importantly, there was a putative miR-21 binding site in exon 5 of GAS5; deletion of the miR-21 binding site abolished this activity. We further showed that biotin labeled GAS5 RNA probe was able to pull down the RNA-induced silencing complex (RISC) and subsequently identified miR-21 in this GAS5-RISC complex, implying that miR-21 and GAS5 may regulate each other in a way similar to the microRNA-mediated silencing of target mRNAs. Together, these results support the “endogenous competitive RNA” concept that microRNAs may regulate not only protein-coding genes, but also non-coding genes such as GAS5.

Citation Format: Ziqiang Zhang, Yin-yuan Mo. Negative regulation of lncRNA GAS5 by miR-21. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2013-1829
Abstract 1875: A novel series of metabolic activators of PKM2 alter oncogene-meditated changes in tumor cell metabolism.
Authors: Bahr Brigham L. , Stevens Jenny , Squire Spencer , Moreno Christopher , Call Lee T. , Stephens Bret J. , Mollard Alexis , Warner Steven L. , Bearss David J.
Published: August 2014

Rather than relying on oxidative phosphorylation for the generation of ATP, human tumor cells primarily utilize aerobic glycolysis to metabolize glucose (the Warburg effect). Pyruvate kinase is a metabolic enzyme that converts phosphoenolpyruvate (PEP) to pyruvate, catalyzing the rate-limiting step of glycolysis. The M1 isoform of pyruvate kinase (PKM1) that is the principal isoform in most adult differentiated tissues, while the PKM2 splice variant is the main isoform in embryonic tissues and in all cancer cells. Unlike the M1 splice form (PKM1), which is found in its tetrameric active form in cells, PKM2 is found in cells as an inactive dimer under normal physiological conditions. Tetramerization of PKM2 requires binding of the allosteric activator fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate, resulting in a fully active enzyme. Regulation of PKM2 activity in cancer cells may allow glycolytic intermediates to be diverted into other biosynthetic pathways necessary for biomass production. PKM2 expression enhances tumorigenicity of cells while PKM1 expression represses it. This suggests that activators of PKM2 may have anti-tumor properties by forcing PKM2 to act more like PKM1. We have a series of small molecule PKM2 activators that exhibit low nM activation activity in biochemical and cell-based assays. These compounds increase pyruvate kinase activity in cancer cells and lead to an increase in pyruvate and ATP production. Our studies show that PKM2 activators inhibit the growth of lung cancer cell lines in vitro and in vivo and can reverse the metabolic changes induced by oncogenes such as k-Ras and c-Myc in lung cancer cells. The current lead compound was tested in established subcutaneously implanted A549 lung adenocarcinoma xenografts, where we observed a statistically significant 54% decrease in tumor growth, with no observable toxicity. These data suggest that this class of PKM2 activators is effective as tumor cell metabolic regulators with anti-tumor activity for lung cancer and potentially other malignancies.

Citation Format: Brigham L. Bahr, Jenny Stevens, Spencer Squire, Christopher Moreno, Lee T. Call, Bret J. Stephens, Alexis Mollard, Steven L. Warner, David J. Bearss. A novel series of metabolic activators of PKM2 alter oncogene-meditated changes in tumor cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1875. doi:10.1158/1538-7445.AM2013-1875
Abstract 1914: Molecular function of the long noncoding RNA SPRY4-IT1 in human melanomas.
Authors: Zhao Wei , Mazar Joseph , Li Jian-Liang , Brill Lawrence M. , Ratnam Maya , Khalil Ahmad M. , Mattick John S. , Dinger Marcel E. , Perera Ranjan J.
Published: August 2014

Recently, we reported that the long noncoding RNA (lncRNA) SPRY4-IT1, which lies within the intronic region of the Sprouty4 gene, is upregulated in human melanomas and influences cell growth, proliferation, and motility, suggesting it may play an important role in the molecular etiology of human melanomas. Here, we report the identification of the full-length of SPRY4-IT1 transcript and its molecular function in human melanoma cells. SPRY4-IT1 transcripts are predominantly found in cytoplasm and accumulate in the polysome fraction of the cell suggests that they may interact with cellular proteins and/or interact with mRNAs to modulate the protein translation. We have identified a group of SPRY4-IT1 interacting proteins by isolating SPRY4-IT1 and protein binding complex and further characterization by mass spectrometry analysis. When SPRY4-IT1 ectopically expressed in melanocytes, it induced formation of multinucleated giant cells and modulates the expression of many genes including a subset of anti-apoptotic, cell cycle regulatory, DNA packaging, chromosome organization and chromatin architecture genes. Notably, the downregulation of the tumor suppressor gene dipeptidyl peptidase IV (DPPIV) and the upregulation the cellular proliferation marker Ki67 indicates the relevance of SPRY4-IT1 in the transition of melanocytes to melanomas.

Citation Format: Wei Zhao, Joseph Mazar, Jian-Liang Li, Lawrence M. Brill, Maya Ratnam, Ahmad M. Khalil, John S. Mattick, Marcel E. Dinger, Ranjan J. Perera. Molecular function of the long noncoding RNA SPRY4-IT1 in human melanomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2013-1914

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2025: Prevention of mammary tumor progression via intraductal injection of nanoparticle-formulated siRNA.
Authors: Krause Silva , Brock Amy , Goldberg Michael , Ingber Donald E.
Published: August 2014

The contribution of specific genes to the development and progression of mammary tumors has been hampered by the inability to silence these genes locally in the mammary gland. Localized intraductal chemotherapy treatments have been explored and offer significant advantages in terms of reduced toxicity and side effects. Therefore, we wanted to explore the possibility of gene knockdown using siRNA delivery via intraductal injection. FVB C3(1)-SV40Tag transgenic female mice spontaneously develop hyperplastic mammary lesions that progress to invasive mammary tumors. Hyperplastic ducts form as early as 12 weeks of age and progress into DCIS-resembling tumors by 16 weeks of age. These further develop into invasive tumors that invade locally into adipose tissue and muscle and occasionally form metastases to lung by 18 to 20 weeks of age. We intraductally injected nanoparticle-formulated siRNA targeted to the SV40 T-antigen transgene beginning at 8 weeks of age through 23 weeks of age into the mammary gland twice a month. As controls, transgenic females were either left untreated or intraductally injected with a non-targeting siRNA. Tumor formation was monitored using ultrasound technology that allows for tumor detection as early as 0.4mm in diameter. At 23 weeks of age, when 100% of the control mice developed tumors, only 1 of 8 mice that received siRNA to the transgene had developed a small tumor. Histological analysis of the mammary glands confirmed development of early lesions but tumor progression was successfully prevented. Mammary lesions were analyzed for proliferation, apoptosis and expression of various markers of differentiation, including hormone receptor status. We showed that siRNA was not detectable in any other organs of the mouse, including the liver. Furthermore, we confirmed that frequent intraductal siRNA injection did not interfere with subsequent pregnancy or lactation, a period in which the mammary gland undergoes extensive remodeling to allow for milk production. Our long-term goal is to leverage the information we uncovered through this effort to target key genes we identified through gene network analysis aiming to prevent the progression of DCIS to invasive tumors. Moreover, this new technology can be utilized to explore the role of genes during specific times during development and tumor formation.

Citation Format: Silva Krause, Amy Brock, Michael Goldberg, Donald E. Ingber. Prevention of mammary tumor progression via intraductal injection of nanoparticle-formulated siRNA. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2013-2025
Abstract 2185: Development and characterization of the fully human chimeric protein GrB-TWEAK targeting Fn14-positive solid tumor cells.
Authors: Zhou Hong , Migliorini Mary , Cao Yu , Cheung Lawrence H. , Hittelman Walter N. , Winkles Jeffrey A. , Rosenblum Michael G.
Published: August 2014

The TNFR superfamily member Fn14, the receptor for TNF-like weak inducer of apoptosis (TWEAK), has emerged as a potentially clinically valuable target for cancer therapy. Specifically, Fn14 expression is relatively low in normal tissues but elevated in various human solid tumor types, including brain, breast, lung and melanoma, and can be a negative prognostic indicator. To target Fn14-overexpressing cancer cells, we constructed via PCR a novel chimeric protein designated granzyme B (GrB)-TWEAK, using the human TWEAK receptor-binding domain as the targeting moiety and the human pro-apoptotic serine protease GrB as the cytotoxic agent. The fusion construct was expressed in mammalian cells and purified to homogeneity from conditioned media. BiaCore analysis of TWEAK and GrB-TWEAK binding to the Fn14 extracellular domain indicated that these proteins bound to Fn14 with similar affinity, with Kds of ∼ 3 nM and ∼ 8 nM, respectively. Confocal immunofluoresence studies showed that GrB-TWEAK specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. GrB-TWEAK activity was assessed using a panel of 26 human cancer cell lines and it demonstrated impressive and selective cytotoxicity to Fn14-expressing cells in the low nanomolar range (IC50 ranged from 0.4 nM-330 nM), which was 6- to 1117-fold more potent than free GrB. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. Mechanistic studies demonstrated that GrB-TWEAK activated caspase cascades and cytochrome C-related pro-apoptotic mechanisms consistent with the known intracellular functions of GrB in target cells. Preliminary mouse xenograft tumor model studies are ongoing. This work was conducted, in part, by the Clayton Foundation for Research (MGR) and supported by NIH grant NS055126 (JAW).

Citation Format: Hong Zhou, Mary Migliorini, Yu Cao, Lawrence H. Cheung, Walter N. Hittelman, Jeffrey A. Winkles, Michael G. Rosenblum. Development and characterization of the fully human chimeric protein GrB-TWEAK targeting Fn14-positive solid tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2185. doi:10.1158/1538-7445.AM2013-2185
Abstract 2348: Discovery of AZD6738, a potent and selective inhibitor with the potential to test the clinical efficacy of ATR kinase inhibition in cancer patients.
Authors: Jones Clifford D. , Blades Kevin , Foote Kevin M. , Guichard Sylvie M. , Jewsbury Philip J. , McGuire Thomas , Nissink Johannes W. , Odedra Rajesh , Tam Kin , Thommes Pia , Turner Paul , Wilkinson Gary , Wood Christine , Yates James W.
Published: August 2014

AZD6738 is a potent and selective inhibitor of ataxia telangiectasia and rad3 related (ATR) kinase with the potential to be used in the clinic. We report the discovery of a morpholino-pyrimidine series and its subsequent optimisation to the pre-clinical candidate AZD6738 which has a good overall balance of potency, selectivity, pharmacokinetic and biopharmaceutical properties suitable for oral dosing. ATR is a serine/threonine protein kinase involved in DNA damage response signalling caused by DNA replication associated stress. Activation of ATR at stalled replication forks leads to suppression of replication fork origin firing, promotes repair and S/G2-cell cycle checkpoints to prevent premature mitosis and maintain genomic integrity. Failure to resolve damage leads to genomic instability and if sufficiently high, cell death. Stalled replication forks may collapse leading to formation of DNA double stranded breaks and activation of the ataxia telangiectasia mutated (ATM) kinase. ATM works in conjunction with ATR to efficiently resolve replication associated DNA damage creating a co-dependency with loss of one leading to a greater reliance on the other to maintain genomic stability. ATM is frequently inactivated across B-cell malignancies, head and neck, breast and lung cancers through chromosomal deletion, promoter hypermethylation or mutation. ATM-deficient tumours are hypothesised to be more reliant on ATR for survival and specific inhibition of ATR may lead enhanced anti-tumour activity while minimizing normal tissue toxicity. AZD6378 has the potential to test the clinical efficacy of ATR inhibition in cancer patients as monotherapy against ATM-deficient tumours or in combination with chemo or radiotherapy.

Citation Format: Clifford D. Jones, Kevin Blades, Kevin M. Foote, Sylvie M. Guichard, Philip J. Jewsbury, Thomas McGuire, Johannes W. Nissink, Rajesh Odedra, Kin Tam, Pia Thommes, Paul Turner, Gary Wilkinson, Christine Wood, James W. Yates. Discovery of AZD6738, a potent and selective inhibitor with the potential to test the clinical efficacy of ATR kinase inhibition in cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2348. doi:10.1158/1538-7445.AM2013-2348
Abstract 247: ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.
Authors: Jiang Qingfei , Crews Leslie A. , Barrett Christian L. , Court-Recart Angela , Goff Daniel , Sadarangani Anil , Rusert Jessica , Morris Sheldon , Goldstein Lawrence , Chun Hye-Jung , Marra Marco , Fraser Kelly , Dao Kim-Hien , Minden Mark , Jamieson Catriona
Published: August 2014

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. While DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity may also be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole transcriptome sequencing of normal, chronic phase (CP) and serially transplantable blast crisis (BC) chronic myeloid leukemia (CML) progenitors revealed increased interferon-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the interferon responsive ADAR1 p150 isoform and a propensity for increased A-to-I RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promoted expression of the myeloid transcription factor PU.1 and induced malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a mis-spliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of BC CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

Citation Format: Qingfei Jiang, Leslie A. Crews, Christian L. Barrett, Angela Court-Recart, Daniel Goff, Anil Sadarangani, Jessica Rusert, Sheldon Morris, Lawrence Goldstein, Hye-Jung Chun, Marco Marra, Kelly Fraser, Kim-Hien Dao, Mark Minden, Catriona Jamieson. ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 247. doi:10.1158/1538-7445.AM2013-247
Abstract 2640A: Cell stiffness as a new indicator of diagnosis for human lung cancer cells and their metastasis.
Authors: Suganuma Masami , Takahashi Atsushi , Watanabe Tatsuro , Akiyama Hirohiko , Nakajima Yuki , Mondal Anupom , Fujiki Hirota
Published: August 2014

Our previous study with atomic force microscopy (AFM) revealed that highly metastatic B16-F10 cells have lower cell stiffness than low metastatic B16-F1 cells, and that treatment of the cells with a green tea polyphenol, EGCG, increased cell stiffness, associated with inhibition of metastasis. Since cell stiffness measured by Young's modulus (Pa) using AFM is attracted the attention as a new indicator of cancer diagnosis, we expanded the study to human lung cancer cell lines, comparing with primary cultured normal bronchial epithelial cells.

We found that cell stiffness of lung cancer cell lines was significantly lower than that of human immortalized peripheral lung epithelial cells (HPL1D): Young's moduli for two adenocarcinoma cell lines (A549 and H322) were 2.4 kPa and 2.2 kPa; that of squamous cell carcinoma cell line (H1703): 0.6 kPa; a large cell carcinoma cell line (H1299): 0.8 kPa; and HPL1D: 4.9 kPa. Furthermore, primary cultured normal bronchial epithelial cells (NBEC) established from ten lung cancer patients showed high Young's moduli from 3.0 kPa to 7.4 kPa. These data indicate that soft cell stiffness is a new indicator of diagnosis for lung cancer. Furthermore, it is important to note that H1703 and H1299 cells with low cell stiffness had high motile activity, compared with A549 and H322 cells with high cell stiffness. This indicates that low cell stiffness of lung cancer cell lines was associated with high motility, suggesting high metastatic ability. Treatments with EGF significantly induced transformation of HPL1D cells with morphological change, resulting in significant reduction of cell stiffness from 3.4 kPa to 1.5 kPa. These data suggest that low cell stiffness is induced in carcinogenic process. A histogram of Young's moduli indicates that a cancer cell line is a conglomerate of various cells with varying cell stiffness. In order to study the change of cell stiffness during cell-cycle, we established H1299/Fucci cells by introducing fluorescent-cell-cycle-indicator (Fucci) gene into H1299 cells. Young's moduli for each cell-cycle phase of H1299/Fucci cells were 1.71 kPa for G1, 1.64 kPa for G1→S, 1.55 kPa for S/G2 and 1.64 kPa for M→G1. Thus, changes in cell stiffness were observed depending on cell-cycle regulation, but the difference was small. Based on all data, we think cell stiffness is a dynamic feature of cancer cells, associated with motility and metastasis of lung cancer.

Citation Format: Masami Suganuma, Atsushi Takahashi, Tatsuro Watanabe, Hirohiko Akiyama, Yuki Nakajima, Anupom Mondal, Hirota Fujiki. Cell stiffness as a new indicator of diagnosis for human lung cancer cells and their metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2640A. doi:10.1158/1538-7445.AM2013-2640A
Abstract 276: Microtentacle stabilization during TGFβ-induced EMT as a new therapeutic target to reduce circulating tumor cell metastasis.
Authors: Slovic Jana , Vitolo Michele I. , Whipple Rebbecca A. , Boggs Amanda E. , Charpentier Monica S. , Bhandary Lekhana , Thompson Keyata , Martin Stuart S.
Published: August 2014

Circulating tumor cells (CTCs) have been identified as a predictive factor of patient outcome and disease progression in breast cancer. Solid tumor metastasis involves detachment of epithelial cells into the lymphatics or vasculature, and the subsequent reattachment in distant tissues to establish new tumors at metastatic sites. Our lab has identified tubulin-based protrusions, termed microtentacles (McTNs), which facilitate tumor cell re-attachment and can penetrate endothelial layers to promote the retention of CTCs in distant tissues. In the current study, we examine the role of the physiological cytokine, TGFβ, in the cytoskeletal changes that promote McTN formation. McTNs are induced during the epithelial-to-mesenchymal transition (EMT) that accompanies tumor progression, and previous studies have shown that TGFβ can promote an EMT in a pathologic setting, despite its normal role as an anti-proliferative factor. When we treated MCF10A nontumorigenic mammary epithelial cells and MCF-7 breast tumor cells with TGFβ, immunoblotting demonstrated changes in protein expression consistent with an EMT, such as increased vimentin and N-Cadherin and decreased E-Cadherin expression. Alongside these typical EMT-induced protein expression changes, levels of detyrosinated α-tubulin (Glu-tubulin) also increased, which is a marker of stabilized microtubules that also promotes McTN formation. Furthermore, immunofluorescence demonstrated that Glu-tubulin was highly diffuse in untreated cells, but became organized into distinct cytoplasmic filaments upon treatment of cells with TGFβ. Detached MCF10A and MCF-7 cells also showed increased McTNs following exposure to TGFβ. Real-time measurement of cell reattachment by electrical impedance demonstrated that the increased McTNs associated with TGFβ treatment resulted in more efficient and faster cell reattachment.

These results emphasize that the post-translational alterations in microtubules promoted by TGFβ can promote increased McTNs and that therapies targeting the molecular mechanisms underlying these TGFβ-induced cytoskeletal changes could provide new targets aimed at reducing CTC reattachment and metastasis.

Citation Format: Jana Slovic, Michele I. Vitolo, Rebbecca A. Whipple, Amanda E. Boggs, Monica S. Charpentier, Lekhana Bhandary, Keyata Thompson, Stuart S. Martin. Microtentacle stabilization during TGFβ-induced EMT as a new therapeutic target to reduce circulating tumor cell metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 276. doi:10.1158/1538-7445.AM2013-276
Abstract 2763: Activity of dasatinib and potential escape mechanisms in diffuse intrinsic pontine glioma (DIPG) models.
Authors: Truffaux Nathalene , Dret Ludivine Le , Puget Stephanie , Vassal Gilles , Geoerger Birgit , Grill Jacques
Published: August 2014

Background

Diffuse intrinsic pontine glioma (DIPG) have a dismal prognosis and their biology is not yet well explored. Using an integrated molecular profiling approach we revealed PDGFRA gene amplification and mutations in the extracellular domain as a characteristic of the oligodendroglial subgroup in newly diagnosed pediatric DIPG. Therefore we explored the therapeutic potential of dasatinib, an inhibitor of the PDGFR, Src, and Ephrin tyrosine-kinases in newly developed DIPG models.

Materials and Methods

A unique panel of 12 DIPG cell lines has been established from fresh tumor material biopsies in newly diagnosed children with DIPG with a 100% success rate. Four were oligoastrocytomas Grade 2 and 3, five astrocytomas Grade 2, 1 GBM and 2 non further specified; two had a gain of the PDGFRA gene. Effects of dasatinib on proliferation, invasion and cytotoxicity in vitro were determined at concentrations of 0.001-1 μM using the Live-cell imaging assay of the IncuCyte system. Cell growth and cell invasion were monitored by confluence algorithm, and cytotoxicity was determined by loss of membrane integrity generating nuclear staining by a fluorescent live-cell imaging reagent. Tyrosine-kinase receptors (RTK) and downstream signaling expression was assessed by phospho-RTK array and Western blot analysis.

Results

Anti-proliferative effects of dasatinib were observed in all 6 cell lines tested with IC50s between 0.02-0.07 μM. This compared favorably with the sensitive adult glioma T98G cell line and the pediatric supratentorial glioma SF188 cell line (0.15 μM and 0.3 μM, respectively). Significant dose-dependent cytotoxic effects were observed in two DIPG cell lines (NEM145 and NEM 168) and anti-invasive effects were seen in all cells at 0.1 μM.

The two most sensitive cell lines exhibited PDGFRA activation and their primary tumors had a gain of PDGFRA. While Src was activated in all cell lines, we noticed that the less sensitive cell lines had MET overexpression.

Conclusion

Primary DIPG cell lines are highly sensitive in vitro to dasatinib suggesting its exploration is children with DIPG. Sensitivity may be linked not exclusively to PDGFRA activation but also to Src activation. The role of MET activation in escape mechanisms is currently explored.

Citation Format: Nathalene Truffaux, Ludivine Le Dret, Stephanie Puget, Gilles Vassal, Birgit Geoerger, Jacques Grill. Activity of dasatinib and potential escape mechanisms in diffuse intrinsic pontine glioma (DIPG) models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2763. doi:10.1158/1538-7445.AM2013-2763
Abstract 2835: Combined stimulation of TLR9 and 4.1BB receptors enhances Trp2 peptide-mediated B16 melanoma regression by increasing Ag-specific CTL activity and infiltration to tumor sites.
Authors: Sin Jeong-Im , Ryoo Hun Mo
Published: August 2014

We previously reported that intratumoral electroporation (IT-EP) with IL-12 cDNA (IT-EP/IL12) led to the eradication of established B16 melanoma tumors in mice. Tumor eradication required the participation of CD8+ T cells, but not CD4+ T cells and NK cells. IT-EP/IL12 induced antigen-specific CD8+ T cell responses against the immunodominant Trp2180-188 epitope and generated a systemic response, resulting in significant therapeutic effects against distal, untreated tumors. In this study, we observed that when Trp2180-188 self-peptides were combined with the TLR9 agonist, CpG-oligodeoxynucleotide (ODN) for antitumor immune therapy, self-tolerance was broken, giving rise to induction of Trp2-specific CTL responses and some antitumor activity against established melanoma (7 mm in mean diameter). A combination of anti-4.1BB antibodies with Trp2 peptides plus CpG-ODN increased the antitumor regression rate from 0 to 75%. This effect was concomitant with greater induction of Ag-specific CD8+ CTLs and their infiltration into the tumor sites, highlighting the importance of combined stimulation of TLR9 and 4.1BB for achieving tumor eradication. These findings may have implications for designing peptide-based therapeutic vaccines for cancer-patients.

Citation Format: Jeong-Im Sin, Hun Mo Ryoo. Combined stimulation of TLR9 and 4.1BB receptors enhances Trp2 peptide-mediated B16 melanoma regression by increasing Ag-specific CTL activity and infiltration to tumor sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2013-2835
Abstract 2929: HIF is necessary, but not sufficient for the hypoxia response.
Authors: Hu Cheng-Jun , Pawlus Matthew , Sena Johnny , Wang Liyi
Published: August 2014

Hypoxia is a common characteristic of many solid tumors. Hypoxia inducible factor HIF1α and HIF2α have been shown to drive malignant progression by activating genes involved in angiogenesis, proliferation, anaerobic metabolism, and other processes that enable tumor cells to survive or to escape their O2-deficient microenvironments. While the major players in the hypoxia-mediated HIFα protein stability and the functions of HIF1α/ARNT and HIF2c/ARNT proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors and how HIF regulates its target genes in the hypoxic transcriptional response are much less understood. We report here that the “Signal Transducer and Activator of Transcription-3” (STAT3) and the basic-helix-loop-helix-leucine-zip transcription factor, upstream stimulatory factor-2 (USF2) are required for the hypoxic transcriptional response, specifically for hypoxic activation of HIF1 and HIF2 target genes respectively. Mechanistically, STAT3 (or USF2) activates HIF1 (or HIF2) target genes by binding to HIF1 (or HIF2) target gene promoters, interacting with HIF1α (or HIF2α) protein and recruiting co-activators CBP and p300 to form enhanceosome complexes that contain HIFα, USF2, CBP, p300 and RNA Pol II on HIF target gene promoters. Interestingly, we found that HIF1α/ARNT and HIF2α/ARNT activate their target genes by recruiting Brm/Brg-1 SWI/SNF complexes to remodel HIF target gene promoter and thus increase HIF target gene activation. These studies established additional players in hypoxic transcriptional response post HIFα protein stabilization by hypoxia. In addition, our studies also showed that co-activators USF2/STAT3 and HIF cooperatively promote the hypoxia response by recurring histone acetylases and chromatin remolding complexes respectively. Uncovering the role of STAT3 and USF2 in the hypoxia response established the foundation to specifically inhibit HIF1 or HIF2 activity for solid tumor treatment.

Citation Format: Cheng-Jun Hu, Matthew Pawlus, Johnny Sena, Liyi Wang. HIF is necessary, but not sufficient for the hypoxia response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2929. doi:10.1158/1538-7445.AM2013-2929

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3020: The gep protooncogene Gα12 mediates LPA-stimulated activation of CREB in ovarian cancer cells.
Authors: Ha Ji Hee , Dhanasekaran Danny , Ward Jeremy , Yang Yoon Mi , Kim Sang Geon , Varadarajalu Lakshmi
Published: August 2014

Lysophosphatidic acid (LPA), plays a critical role in the pathophysiology of ovarian cancers. Our previous studies have shown that LPA stimulates the proliferation of ovarian cancer cells through GNA12, the gep protooncogene Gα12. The present study is focused on identifying the Gα12-dependent mechanism through which LPA stimulates the proliferation of ovarian cancer cells. Using ovarian cancer cells in which the expression of Gα12 is silenced, we demonstrate here that Gα12-dependent mitogenic signaling by LPA involves the atypical activation of the transcription factor, cAMP-response element binding protein (CREB). Protein/DNA array analyses using LPA-stimulated Hey8 cells in which the expression of Gα12 was silenced indicated the potent but Gα12-dependent activation of CREB by LPA. After validating the array data with immunoblot analysis using antibodies specific to Ser133-phosphorylated active form of CREB, we demonstrate that the expression of the constitutively activated mutant of Gα12 stimulate the activation of CREB even in the absence of LPA whereas silencing of Gα12 abrogates such LPA-stimulated activation in multiple ovarian cancer cell lines. Furthermore, our results indicate that the robust activation of CREB by LPA is an early event, which can be monitored upon LPA stimulation from 3 minutes onwards. In addition, we establish that LPA/Gα12-dependent activation of CREB involves an atypical cAMP-independent mechanism involving Ras and ERK. More significantly, our findings indicate that the expression of the dominant negative mutant of CREB (S133A) leads to a reduction in LPA-stimulated increase in Cyclin A levels along with an attenuation of LPA-stimulated proliferation of these cells. Thus, these studies unravel a novel Gα12-dependent mechanism involving CREB through which LPA stimulates the proliferation of ovarian cancer cells.

Citation Format: Ji Hee Ha, Danny Dhanasekaran, Jeremy Ward, Yoon Mi Yang, Sang Geon Kim, Lakshmi Varadarajalu. The gep protooncogene Gα12 mediates LPA-stimulated activation of CREB in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2013-3020
Abstract 3031: The receptor tyrosine kinase layer of breast cancer cell lines is predictive of the response to therapeutic drugs.
Authors: Niepel Mario , Hafner Marc A. , Pace Emily A. , Schoeberl Birgit , Sorger Peter K.
Published: August 2014

Currently, clinicians use the levels of the hormone receptors for estrogen (ER) and progesterone (PR) and amplifications of the ErbB2 receptor tyrosine kinase (RTK) to distinguish three groups of breast cancers and guide the choice of therapeutic treatments. In this work we show that a limited number of measurements characterizing breast cancer cell lines at the level of RTKs can be used to predict cell line sensitivity to therapeutic inhibitors.

We chose a panel of approximately 40 breast cancer cell lines, treated them with 22 different growth factors and cytokines at a saturating and subsaturating dose for 10, 30, and 90 min and measured the phosphorylation of key kinases downstream of the receptors. In addition, we measured the expression and phosphorylation levels of approximately 20 RTKs under serum starvation conditions. We used the measurements to predict the GI50 values of the cell lines in response to over 45 therapeutic inhibitors by partial least square regression. We found that our measurements are sufficient to predict the response to about half of the drugs studied. The majority of drugs we are able to predict target RTKs directly or kinases immediately downstream of the receptors. In some cases we are able to predict the response to drugs that target processes seemingly unrelated to our measurements. We are currently exploring the data to uncover what drives sensitivity and resistance to drugs, gain mechanistic insight in the action of cancer therapeutics, and find predictors of drug response that may be useful in a clinical setting.

Our results suggest that a carefully selected set of measurements might be sufficient to classify responsiveness of breast cancers to a number of cancer drugs currently used in the clinic. This is particularly relevant in the case of cancers like triple negative breast cancers for which choosing effective treatment has proven difficult.

Citation Format: Mario Niepel, Marc A. Hafner, Emily A. Pace, Birgit Schoeberl, Peter K. Sorger. The receptor tyrosine kinase layer of breast cancer cell lines is predictive of the response to therapeutic drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3031. doi:10.1158/1538-7445.AM2013-3031