bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 28: Resequence-analysis of 166kb KITLG region in eamilial testicular germ cell tumors
Authors: Chung Charles C. , Yeager Meredith , Boland Joseph , Greene Mark , Chanock Stephen , Kratz Christian
Published: June 2012

Recent genome-wide association studies (GWAS) have identified multiple loci associated with testicular germ cell tumor (TGCT) risk, particularly in KITLG on chromosome 12q22, which has the strongest per-allele odds ratio in cancer GWAS to date. KITLG, also known as stem cell factor or Steel, encodes the ligand for the membrane receptor tyrosine kinase, c-KIT. It is notable that mutations in the KIT/KITLG system have been implicated in both testicular cancer development and infertility, both of which are established TGCT risk factors. Therefore, a comprehensive cataloguing of variants around the region is essential for further functional studies. We defined a 166kbps region (chromosome 12: 87371132-87537273) around KITLG, based on linkage disequilibrium patterns and tag analysis using HapMap III CEU and the 1000 genome CEU low-coverage pilot data (July 2010 release). No obvious recombination hotspot was observed within a 1Mb region centered on KITLG. Our study sample set included 10 parent-son trios, of which 6 trios contained 2 familial TGCT cases, 51 familial TGCT cases and 36 sporadic bilateral cases, totaling 117 individuals, all from the National Cancer Institute's Familial Testicular Cancer Study (FTCS). We have catalogued common and rare KITLG variants using deep coverage in next-generation sequencing and also identified haplotypes possibly enriched in familial cases in contrast to normal controls. These study results will facilitate further functional elucidation of TGCT susceptibility loci within this genomic region.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 28. doi:10.1158/1538-7445.AM2011-28
Abstract 2801: Fueling immunity: The role of Myc in metabolic reprogramming of activated T cells
Authors: Wang Ruoning , Dillon Christopher , Green Douglas
Published: June 2012

One of the fundamental problems in animal cells is the coordination of the metabolic program with cell proliferation-associated bioenergetic demand. Primary T lymphocytes rapidly switch from a quiescent phase to a rapid-proliferation phase one day after antigen stimulation. To meet the bioenergetic and biosynthetic demand for this burst of proliferation, T cells rapidly reprogram their metabolic pathways from fatty acid beta-oxidation and glucose aerobic respiration to glycolysis (converting glucose to lactate) and glutaminolysis (converting glutamine to metabolites of the TCA cycle). This is reminiscent of tumor-associated metabolic reprogramming in many ways. Recent work has indicated that oncogenic signaling can reform tumor metabolic programs by targeting relatively few key metabolic genes. However, we found that global regulation of the metabolic gene transcriptome precedes the switch of T cell metabolic activities. In silico analysis of DNA-binding motifs associated with the metabolic gene expression profile revealed HIF1a, c-Myc and p53 as three of the top-ranked candidate transcription factors responsible for T cell metabolic reprogramming. Consistent with this, HIF1a, c-Myc and p53 are each highly induced in primary T cells upon antigen stimulation and have been implicated in the regulation of tumor metabolism. However, while germline deletion of p53 and acute deletion of HIF1a showed no defects in metabolic reprogramming upon antigen stimulation, acute deletion of c-Myc in mature T lymphocytes profoundly reduced antigen-stimulated glycolysis and glutamine oxidation in primary T cells. In addition, c-Myc deletion significantly compromised T cell growth and T cell proliferation after antigen stimulation. Finally, we found that many of the key metabolic genes involved in glycolysis and glutamine oxidation are regulated in a c-Myc-dependent manner. Our results further suggest a novel c-Myc dependent metabolic pathway linking glutamine oxidation to the biosynthesis of polyamines, which is a group of metabolites essential for cell proliferation. Our studies clearly demonstrate that primary T lymphocytes drive a c-Myc-dependent metabolic reprogramming upon antigen stimulation, which may represent a general mechanism of the metabolic reprogramming under both pathological and physiological conditions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2801. doi:10.1158/1538-7445.AM2011-2801
Abstract 2837: Adiponectin knockout enhances intestinal carcinogenesis in Min and wild-type mice
Authors: Mutoh Michihiro , Teraoka Naoya , Takasu Shinji , Takahashi Mami , Onuma Kunishige , Kubota Naoto , Kadowaki Takashi , Wakabayashi Keiji , Nakagama Hitoshi
Published: June 2012

Obesity-associated cancers, including colon cancer, are increasing in Western countries. It has been demonstrated that increased amounts of visceral fat and decreased levels of plasma adiponectin (APN) are associated with development of human colorectal cancer. Thus, we here investigated the function of APN in intestinal carcinogenesis. Male APN+/+, APN+/- or APN-/- C57BL/6J mice were injected with azoxymethane (AOM) once a week for 6 weeks, which led to the development of intestinal tumors. C57BL/6J mice with each genotype were sacrificed at the age of 55 weeks, and the number and size of colorectal tumors were examined by pathological study. Furthermore, these strains of mice were also crossed with Min mice (C57BL/6J background) to assess the effects of APN on intestinal polyp formation. At the age of 9 and 12 weeks, Min mice each with the APN genotype were sacrificed, and the number and size of intestinal polyps were examined by pathological study. Levels of adipocytokine and phosphorylation of AMPK were also evaluated to clarify the effect of APN on intestinal tumor development. The incidence of AOM-induced tumors was 40% of APN+/+, 50% of APN+/- and 71% (p<0.05) of male APN-/- C57BL/6J mice, respectively. Multiplicities of AOM-induced tumors in each genotype were 0.5±0.7, 0.6±0.7 and 1.1±1.0 (p<0.05), respectively. The total number of intestinal polyps developed in 9-week-old male APN+/- Min and APN-/-Min mice increased 2.4- and 3.2-fold compared with those in APN+/+Min mice, respectively, and was further enhanced at 12 weeks of age, being 3.2- and 3.4-fold, respectively. Studies with female mice gave similar results. Phosphorylation levels of AMPK in intestinal epithelial cells were decreased in APN-/- mice compared with APN+/+ mice. Among serum adipocytokines, levels of serum Pai-1 increased in APN-/- C57BL/6J mice and APN-/-Min mice with AOM injection. Activation of AMPK by metformin treatment suppressed Pai-1 expression in Min mice. AOM-induced colorectal aberrant crypt foci in APN-/- C57BL/6J mice were decreased by a Pai-1 blocker. In conclusion, APN-deficient mice developed more intestinal tumors. Decreased phosphorylation of AMPK and increased levels of Pai-1 were observed in APN-deficient mice compared with wild-type mice. APN and its receptors might be good targets for developing colorectal cancer chemopreventive agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2837. doi:10.1158/1538-7445.AM2011-2837
Abstract 2855: The codon 72 polymorphism of p53 influences the transcription of genes involved in immunity and inflammation
Authors: Frank Amanda , Hollstein Monica , Murphy Maureen E.
Published: June 2012

A common polymorphism at codon 72 in the p53 tumor suppressor protein encodes either proline (P72) or arginine (R72). Several groups have reported that in engineered cell lines this polymorphism influences p53's transcriptional, growth arrest, and apoptotic functions. However, the impact of this polymorphism within the context of a living organism has never been examined. In efforts to understand the biological consequences of the p53 codon 72 polymorphism in a living organism, we generated P72 and R72 mice in the background of the Humanized p53 knock-in (Hupki) mouse. In Hupki mice that are extensively back-crossed into a C57Bl/6 background, we find that there are consistent tissue-specific differences in apoptotic function between the P72 and R72 variants. Within the thymus, we find that the P72 variant is associated with increased apoptosis following gamma radiation. Micro-array analysis indicates that a small subset of genes show increased transactivation in thymocytes from P72 mice compared to R72. These genes include caspase 4/11, which mediates the processing of caspase 1. Interestingly, several of these genes are also regulated by NF-kB, and play roles in immunity and inflammation. Mechanistically we show that p53 interacts with the p65 Rel A subunit of NF-kB, and further that the P72 variant of p53 interacts significantly better than R72. These data indicate that the codon 72 polymorphism affects the ability of p53 to interact with NF-kB and to cooperate in the transactivation of genes involved in immunity and inflammation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2855. doi:10.1158/1538-7445.AM2011-2855
Abstract 2862: Bioengineered oncolytic adenovirus induces autophagic cell death through an E2F1-microRNA-7-epidermal growth factor receptor axis
Authors: Tazawa Hiroshi , Yano Shuya , Yoshida Ryosuke , Urata Yasuo , Fujiwara Toshiyoshi
Published: June 2012

Autophagy is known to have a cytoprotective role under various cellular stresses, such as deprivation of nutrients, hypoxia and interruption of growth signaling. In contrast, autophagy also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Recent accumulating evidences have suggested the implication of autophagy on the oncolytic adenovirus-mediated cell death in human cancer cells. However, the molecular mechanism underlying oncolytic adenovirus-mediated autophagic cell death remains unclear. We previously generated a genetically engineered oncolytic adenovirus (Telomelysin®: OBP-301) that replicates and induces autophagic cell death against human cancer cells in a telomerase-dependent manner. As microRNA (miRNA), a post-transcriptional regulator of gene expression, has been recently shown to regulate diverse cell fates including autophagy, we hypothesized that OBP-301 infection induces autophagic cell death through miRNA modulation in human cancer cells. In this study, we attempted the role of miRNA on the OBP-301-mediated autophagic cell death in human cancer cells. We used 3 human cancer cell lines (H1299, A549 and T.Tn), that showed different sensitivities to OBP-301. OBP-301 infection upregulated miR-7 expression in OBP-301-sensitive human cancer cell lines (H1299 and A549), but not OBP-301-resistant human cancer cell line (T.Tn). Expression levels of miR-7 upregulated after OBP-301 infection was significantly associated with OBP-301-mediated cytopathic activity and autophagic cell death, which was confirmed by western blot for autophagy-related proteins (LC3-I/II, Atg5 and p62). We also revealed that OBP-301-mediated miR-7 upregulation mainly depended on enhanced expression of the E2F1 protein after OBP-301 infection. Furthermore, ectopic expression of miR-7 in human cancer cell lines suppressed cell viability and subsequently induced autophagy through inhibition of epidermal growth factor receptor (EGFR) expression. OBP-301 infection efficiently suppressed EGFR expression, which was significantly associated with miR-7 upregulation and cytopathic activity, in human cancer cell lines. Our results suggest that oncolytic adenovirus OBP-301 induces autophagic cell death through an E2F1-miR-7-EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2862. doi:10.1158/1538-7445.AM2011-2862
Abstract 2866: Targeting autophagy in pancreatic cancer
Authors: Ustach Carolyn V. , Bisanz Kristen , Von Hoff Daniel D. , Xu Eric , Han Haiyong
Published: June 2012

Autophagy is a cell survival pathway that has been shown to be activated in a variety of cancers including pancreatic cancer. It has been suggested that cancer cells activate autophagy to survive microenvironmental stresses such as depletion of nutrients, hypoxia and chemotherapeutic agents. Studies have shown that inhibition of autophagy can sensitize cancer cells to the treatment of therapeutic agents. Chloroquine (CQ) is a well established pharmacological inhibitor of autophagy and has been shown to enhance the sensitivity of cancer cells to treatment of cytotoxic agents. Dexamethasone (DEX), a synthetic gluococorticoid, which has been reported to sensitize pancreatic cancer cells to the treatment of gemcitabine (GEM) and carboplatin, is known to induce autophagy. In addition, activation of glucocorticoid receptor (GR) by DEX inhibits NF-kB and AP-1, two activators of survival pathways in many tumors. Whether DEX increases the cytotoxic effects of GEM through autophagy, NF-kB signaling, or a combo of both is unclear. Our preliminary studies show that DEX dramatically enhances the anti-tumor activity of CQ, indicating that the inhibition of autophagy may increase cell sensitivity to drug treatment. Therefore, we hypothesize that a combination pretreatment with CQ and DEX will significantly enhance the anti-tumor activity of GEM in pancreatic cancer, and that such enhancement in GEM activity is induced by the synergistic inhibition of autophagy by CQ and DEX. In vitro cell growth assay results showed that addition of CQ and DEX significantly increase the antitumor activity of GEM in MIA PaCa-2 and SU.86.86 pancreatic cancer cells. Interestingly, combination treatment of DEX+GEM or CQ+GEM did not show significant synergistic effect. A correlation between autophagy inhibition and cell death was evident when levels of LC3B-II protein, a common indicator of autophagy induction, were compared in cells exposed to single or combination drug treatments. In MIA PaCa-2 cells, CQ+DEX with 100nM GEM reduced LC3B-II levels by at least 50%. Animal studies are underway to determine the efficacy of the triple drug combination in vivo. Our results indicate pancreatic cells use autophagy as a cell survival mechanism in response to gemcitabine, and the drug combination dexamethasone and chloroquine are able to inhibit authophagy, thus rendering the pancreatic cancer cells more sensitive to gemcitabine.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2866. doi:10.1158/1538-7445.AM2011-2866
Abstract 2876: A novel multi-parametric assay of cell death pathways: Autophagy, apoptosis, and necrosis
Authors: Shen Dee , Chan Eric , Pande Praveen , Patton Wayne F.
Published: June 2012

Cell death can be classified into a number of types including apoptotic, autophagic, necrotic and others. The hallmarks of apoptotic cell death include exposure of phosphatidyl serine on the extracellular face of the plasma membrane, activation of caspases, disruption of mitochondrial membrane potential, cell shrinkage, DNA fragmentation and DNA condensation. Necrotic cell death is characterized by cell swelling, as well as destruction of the plasma membrane and subcellular organelles, without nuclear fragmentation and condensation. Autophagic cell death is a degradative process involving sequestration of cellular components in double-membrane vesicles that fuse with lysosomes to form the autophagolysosomes. Autophagy is a survival strategy in situations of stress, but can also lead to non-apoptotic cell death. Due to the overall complexity of the cited death pathways, a sensitive assay capable of distinguishing between autophagy, apoptosis and necrosis is fundamental to further understanding of these pathophysiological processes. Thus, a three-parameter cell-based assay was developed utilizing a green-emitting fluorophore to highlight the various vacuolar components of the autophagy pathway, a violet-excitable fluorophore conjugate of annexin V to measure phosphatidyl serine exposure in early stage apoptosis, and a cell-impermeable red fluorescent DNA intercalation dye to measure membrane disintegration in late-stage apoptosis/necrosis. The assay was validated using a range of conditions known to modulate autophagy, apoptosis and necrosis pathways. The assay provides a rapid, information-rich read-out of the various cell death pathways by fluorescence microscopy and flow cytometry and should facilitate better drug activity profiling and clearer kinetic analysis of these fundamental processes in living cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2876. doi:10.1158/1538-7445.AM2011-2876
Abstract 2878: A sensitive cell signaling assay for low level activation of endogenous receptors – a comparison of AlphaScreen® SureFire® and TR-FRET assays
Authors: Hurt Stephen , Titus David
Published: June 2012

Two closely related proteins, ERK1 and ERK2, belong to a widely conserved family of serine/threonine protein kinases whose activities are regulated by phosphorylation. These kinases are components of signal transduction pathways which control critical cellular processes such as growth, differentiation, metabolism and apoptosis. In addition to normal cellular processes, these pathways have been associated with signaling in many cancers. Phosphorylation of ERK 1/2 has been shown to be mediated by signal transduction pathways which are activated by ligand binding to both receptor tyrosine kinase receptors (RTKs) and G protein-coupled receptors (GPCRs). The focus of cell signaling studies is increasingly shifting to endogenous cell surface receptors rather than recombinant, over-expressed receptors. The hope is that these model systems will have greater biological relevance, more closely reflecting the physiology of the organism studied. In such studies the sensitivity of the assay platform becomes critical for success, especially when the endogenous receptors are activated at low levels. Several technologies have been used to quantitate relative levels of protein phosphorylation, including western blot, time-resolved fluorescence resonance energy transfer (TR-FRET), and AlphaScreen® SureFire® assays. AlphaScreen® SureFire® assays are homogenous, bead-based assays designed to measure endogenous levels of intracellular phosphorylated proteins. Here we describe the performance of the AlphaScreen® SureFire® p-ERK 1/2 assay compared to that of a TR-FRET assay. For this study we used two different cell lines. The first was CHO-K1 cells stably transfected with the human somatostatin sst5 GPCR, which is stimulated by its natural ligand somatostatin-28. The second was wild-type HEK 293 cells, which express endogenous receptors for epidermal growth factor (an RTK), and endogenous GPCRs activated by acetylcholine and ATP. In general, the AlphaScreen® SureFire® assay gave higher S/B ratios than the TR-FRET assay in all conditions tested, which in turn can have a significant impact on the assay robustness. In situations that gave wide response windows, such as stimulation of the CHO over-expressed somatostatin receptor or the HEK 293 endogenous EGF receptor, both assay kits yielded excellent results. However, the HEK 293 endogenous GPCRs for acetylcholine and ATP are coupled to a lower production of p-ERK, and in those cases the AlphaScreen® SureFire® assay proved to be much more robust, with significantly higher overall signal levels and S/B ratios.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2011-2878
Abstract 2880: Role of Akt isoforms in chemoresistance of endometrial carcinoma cells
Authors: Lafleur Marie-Judith , Girouard Julie , Asselin Eric
Published: June 2012

Akt (PKB) is a crucial survival kinase that is normally activated by a variety of growth factors. In tumours, its upstream regulation is affected by oncogenic events, which leads to the constitutive activation of the protein. Since studies have demonstrated that the three Akt isoforms exhibit different physiological functions, our hypothesis is that Akt isoforms may contribute differently in chemoresistance of endometrial carcinoma cells. The aim of this study is to determine the effect of a loss of function of each of Akt isoform in chemoresistant KLE endometrial carcinoma cells. In order to determine the specific role of each Akt isoform in cell survival/resistance to apoptosis and proliferation, we stably transfected KLE cells, which normally express Akt1, Akt2 and Akt3, with specific shRNA for each Akt isoform. We treated Akt isoform-specific transfectants for a period of 48 hour with different chemotherapeutics drugs and we performed western blot analysis for pro-apoptotic and anti-apoptotic factors. In this study, we have demonstrated that Akt1 and Akt2 downregulation sensitize KLE cells to cisplatin, doxorubicin and taxol. After treatment of KLE transfectants with all three drugs, we were able to see that activation pro-apoptotic factors such as cleaved caspase-3, -6 and -9 as well as cleaved PARP were induced. Moreover, the expression of anti-apoptotic factor XIAP was downregulated after drug treatment in these cells. Finally, Bcl-2 expression was inhibited after cisplatin treatment in Akt2 negative cells. We also performed MTT proliferation assay after a 72 hours treatment with chemotherapeutic drugs. Results gained from this study show that each Akt isoform is specifically implicated in cell survival. Akt1 and Akt2 shRNA clones were more sensitive to cisplatin, doxorubicin and taxol treatment. Akt3 shRNA clones were also more sensitive to cisplatin and doxorubicin but were less affected by taxol treatment. Our findings highlight the contribution of Akt isoforms in the molecular mechanisms that confer KLE cells resistance to chemotherapeutics drugs-induced apoptosis. According to our hypothesis, results show the differential implication of each Akt isoform in cell resistance to different chemotherapeutics drugs. We also showed that each isoform have a distinct impact on resistance to apoptosis induced by a given drug. Thus, the results confirm that Akt isoforms could be putative targets for therapy of endometrial cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2880. doi:10.1158/1538-7445.AM2011-2880
Abstract 2918: Combining large scale biochemical and cellular profiling data to better understand the mechanism of anti-tumor drugs
Authors: Nauman Sharon , Bode Jacob , Hadden Clare , Byers Lynn , Armbruster Blaine N.
Published: June 2012

This study was designed to elucidate the selectivity and cellular behavior of several marketed kinase inhibitors used in the treatment of cancer patients. Generally, kinase inhibitors are optimized to inhibit a single cellular target associated with cancer progression. However, most currently marketed kinase inhibitors interact with a highly conserved ATP binding site raising the question of their selectivity and therapeutic mechanism of action. Through EMD-Millipore's KinaseProfiler(TM) service, we functionally profiled the selectivity of Imatinib, Sunitinib, Lapatinib as well as several other marketed kinase inhibitors against a panel of over 250 kinases. Having identified the in vitro targets for these inhibitors, we next examined the potential of these small molecules to either directly inhibit several of the identified targets, or indirectly inhibit the activation of key signaling molecules downstream of these targets in several cell lines using EMD-Millipore's SignalProfiler(TM) service. Kinase profiling studies revealed that no kinase inhibitor was selective for a single target, with most inhibitors interacting with ten or more (sometimes phylogenetically distinct kinases) when tested at 1 µM. Additionally, several inhibitors were also found to either inhibit or activate certain GPCRs. The polypharmacology of the kinase inhibitors found through these studies would suggest that interaction with several molecular targets could contribute to either the therapeutic or adverse effects associated with these drugs. Current studies are underway to evaluate the cellular effect of these compounds to 1) resolve whether the compounds’ potential promiscuity, as revealed via biochemical studies, translates to a cellular context and 2) to assess whether drugs that share key therapeutic targets differentially affect cell signaling, due to differences in their off-target interactions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2918. doi:10.1158/1538-7445.AM2011-2918
Abstract 2942: EYA promotes proliferation through up-regulation of cyclin D1
Authors: Li Zhaoming , Hu Junbo , Sun Yue , Li Sean , Pestell Richard G. , Wu Kongming
Published: June 2012

In Drosophila, eye development is coordinately regulated by the Retinal Determination Gene Network (RDGN), mainly consisting of dachshund (dac), eyes absent (eya), and sin oculis (so). The mammalian homologues are Dach, Eya, and Six, respectively. RDGN, although best known for its role in eye specification, is essential for the development of many organs including retinal, kidney, brain, and limb in both flies and mammals. Molecular mechanism studies support a model that Six functions as a DNA-binding factor and Dach/Eya are transcription co-factors. Eya was first characterized as a nuclear factor, interacting with Six and Dach to regulate gene expression in a context-dependent manner. Eya encodes intrinsic protein tyrosine phosphatase activity. Recently, altered expression of the DACH1/Eya/Six complex has been observed in human tumors. EYA and SIX genes are up-regulated and DACH genes are down-regulated. EYA expression was enhanced in breast, ovary and malignant peripheral nerve sheath tumors. To elucidate the function of Eya in human breast cancer and identify the down-stream targets, we established several human breast cancer cell lines (SKBr3, MDA-MB-453 and BT474) stably expressing Eya1 or phosphatase-defective mutant Eya1D327A. In all three cell lines, expression of Eya1 promoted contact-dependent and contact-independent (soft agar assay) growth. MTT assays and growth curves (cell counting) demonstrated enhanced cellular proliferation upon expression of Eya1. In contrast, cells expressing phosphatase-defective mutant Eya1D327A repressed growth were observed. Western blot analysis demonstrated increased abundance of cell Cyclin D1 in EYA1 expressing cells. Quantitative RT-PCR demonstrated about 3-fold increase of cyclin D1 mRNA abundance in MDA-MB453 cells stably expressing wild type Eya1, but not phosphatase-defective EYA1 mutant. EYA1 induced the human cyclin D1 promoter activity evaluated by luciferase assay and the EYA1 phosphatase activity was required. Immunoprecipitation studies demonstrated association of Eya1 with endogenous Six1. As EYA does not have DNA binding ability and SIX1 is known to binds to the cyclin D1 promoter, we hypothesized model in which EYA1 up-regulates SIX1, and thus a EYA1/SIX1 complex activates Cyclin D1. Lentiviral shRNAs targeting human SIX1 or cyclin D1 were transduced into EYA1 over-expressing cells respectively. The growth curve, MTT assays and clone formation results indicated the pro-proliferate effect of EYA1 was abolished by knock down the expression of either cyclin D1 or Six1. Cyclin D1 abundance was reduced by shSIX1. Taken together, our study demonstrated that cyclin D1 is a key target of EYA1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2942. doi:10.1158/1538-7445.AM2011-2942
Abstract 2964: AZD7762-mediated gemcitabine sensitization does not require G2-M checkpoint abrogation in pancreatic cancer cells
Authors: Parsels Leslie A. , Tanska Daria M. , Parsels Joshua D. , Zabludoff Sonya D. , Brown Jeffrey L. , Morgan Meredith A. , Lawrence Theodore S. , Maybaum Jonathan
Published: June 2012

Chk1 inhibition is a promising approach to sensitize pancreatic cancer to gemcitabine (Gem), the current standard therapy for both local control and metastatic disease. One hallmark and proposed biomarker of Chk1 inhibition is abrogation of the DNA damage-induced G2-M checkpoint and aberrant mitotic entry. It is unclear, however, whether this effect is necessary for chemosensitization. In this study, we tested the hypothesis that G2-M checkpoint abrogation is required for Gem sensitization in cells treated with the Chk inhibitor AZD7762.

We first evaluated the ability of AZD7762 to abrogate the G2-M checkpoint in 6 pancreatic cancer cell lines. While AZD7762 sensitized each cell line to a moderately toxic concentration of Gem, G2-M checkpoint abrogation was only observed in 4 of the lines; AsPC1, MiaPaCa2, M-Panc96 and Panc1. Furthermore, only MiaPaCa2 and Panc1 cells underwent premature mitotic entry when treated with Gem + AZD7762. We next tested the hypothesis that G2-M checkpoint abrogation was responsible for AZD7762-mediated Gem sensitization by using the Cdk inhibitors roscovitine and purvalanol A to preserve the checkpoint in the presence of AZD7762 by directly inhibiting cyclinB-cdk1 and mitotic entry. While both Cdk inhibitors blocked mitosis in cells treated with Gem + AZD7762, the effects on chemosensitization varied. Only in AsPC1 and Panc1 cells did roscovitine prevent both G2-M checkpoint abrogation and Gem sensitization. Furthermore, cyclin B1 siRNA prevented AZD7762-mediated G2-M checkpoint abrogation in MiaPaCa2 and Panc1 cells, but did not prevent chemosensitization, suggesting AZD7762-mediated Gem sensitization does not require G2-M checkpoint abrogation.

In addition to abrogating the Gem-induced G2-M checkpoint, AZD7762 promotes S-phase progression. Initial studies suggest AZD7762 may sensitize cells to Gem by abrogating the S-phase checkpoint and permitting S-phase progression in the presence of Gem-induced DNA damage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2011-2964
Abstract 2970: Interference with DNA repair through inhibition of Wee1
Authors: Krajewska Malgorzata , Bisselink Yvette , van Vugt Marcel A.
Published: June 2012

In response to DNA damage, the so-called ‘DNA damage response’ provokes a cell cycle arrest to facilitate DNA repair. Recently, it has become clear that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, the mitotic kinases Cdk1 and Plk1 were shown to be responsible for the inactivation of key DNA damage checkpoint proteins, including Claspin and 53BP1.

Here we have addressed the possibilities to exploit the mitotic inactivation of the DNA damage response with the purpose to block proper cellular responses to DNA damage during interphase. We hypothesized that mimicking the ‘mitotic’ state of the DNA damage response could potentially inhibit DNA repair and could sensitize cancer cells to genotoxic therapy. To this end, we employed chemical inhibition of the Wee1 kinase. Wee1 is known to prevent unscheduled Cdk1 activation and inhibition of Wee1 has previously been shown to provoke Cdk1 activity.

We could show that chemical Wee1 inhibition indeed leads to upregulation of Cdk1 activity. Importantly, we furthermore showed that recruitment of critical repair factors, such as BRCA1 were no longer recruited to sites of DNA damage. Furthermore, we show that DNA repair kinetics were decreased after inhibition of Wee1. Finally, we could show that inhibition of Wee1 sensitized cells to genotoxic chemotheapeutics and irradiation. Thus, forced activation of mitotic kinase interferes with normal DNA damage responses and can be used for radio/chemosensitization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2011-2970
Abstract 2983: Sgo1 haploinsufficiency contributes to enhanced colon carcinogenesis in mice and yet associated with high colonic epithelial apoptosis
Authors: Yamada Hiroshi Y. , Zhang Yuting , Dai Wei , Rao Chinthalapally V.
Published: June 2012

Inactivation of Shugoshin-1 (Sgo1) function or depletion of Sgo1 in mammalian cells causes missegregation of chromosome. Sgo1 plays an obligate role in chromosome cohesion and its defect is implicated in high chromosome instability. Recent studies indicate that Sgo1 levels were significantly decreased in human colonic tumors, suggesting a role in colon carcinogenesis. Here, we report for first time that depletion of Sgo1 lead to the enhanced colorectal carcinogenesis in mice. To understand the role of Sgo1 in colorectal cancer, we have established haploinsufficient Sgo1+/- mouse model. We tested whether Sgo1 deficiency would enhance the colon carcinogenesis in animal exposed to colonic specific carcinogen. At eight weeks of age mice Sgo1+/+ and Sgo1+/- were treated with azoxymethane (AOM), s.c., 4 mg/kg BW, twice weekly for four weeks. Upon treatment with AOM, we analyzed for colonic crypt cell-cycle modifications and later stage colonic ACF and tumor formation. Sgo1-/+ mice showed higher acute cell death rate than Sgo1+/+ mice soon after the AOM-treatment. However, at the endpoint, the number of aberrant crypt foci [Sgo1+/+, 4±1.41 vs Sgo1+/- 12.2 ±1.5, mean ±SD, p<0.0002] and multiplicity of aberrant crypts/foci [Sgo1+/+ 0.4±0.32 vs Sgo1+/- 3.8 ±0.57, mean ±SD, p<0.0001]. Importantly, mean number of colonic microadenoma were 5-fold higher in haploinsufficient mice [Sgo1+/+, 0.8 ±0.45 vs Sgo1+/- 5.0 ± 1.2, p<0001]. Normal-looking colonic mucosa in AOM-treated Sgo1-/+ mice expressed higher Bcl2, p53 and COX-2 proteins compared with Sgo1+/+ type. These results suggest the notion that defect in mitotic machinery can serve both oncogenic and enhance cell death by apoptosis in an environment-dependent manner; Sgo1-/+ mice may be latently antagonizing carcinogenesis by removing damaged cells in higher rate with cell death. Our results for the first time provide functional evidence of Sgo1 in enhancement of precancerous lesions, and their progression to colonic tumors. (Supported by R01-CA-113349 and R01-CA-102942)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2983. doi:10.1158/1538-7445.AM2011-2983
Abstract 3027: SIRT1 promotes N-Myc oncogenesis stabilizing N-Myc protein
Authors: Marshall Glenn M. , Liu Pei Y. , Gherardi Samuele , Scarlett Christopher J. , Bedalov Antonio , Xu Ning , Iraci Nunzio , MacKenzie Karen L. , Haber Michelle , Norris Murray D. , Biankin Andrew V. , Perini Giovanni , Liu Tao
Published: June 2012

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting low-level to high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62 and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor, Cambinol, reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc-induced neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2011-3027
Abstract 3039: Identification of a novel biomarker of resistance to lapatinib common to both breast and oesophagogastric cancer
Authors: Chong Irene , Wetterskog Daniel , Shiu Kai-Keen , Meijer Titia , Natrajan Rachael , Maryou Lambros , Reis-Filho Jorge , Lord Lord J. , David Cunningham , Ashworth Alan
Published: June 2012

HER2 is amplified and/or overexpressed in 15% and 20% of breast and oesophagogastric (OG) cancers, respectively. Results from the recently published ToGA trial reveal that trastuzumab plus standard chemotherapy improves survival in patients with advanced HER2-positive OG cancer compared with chemotherapy alone, which mirrors the survival benefit shown with trastuzumab plus chemotherapy in metastatic breast cancer. The clinical value of lapatinib, already confirmed in advanced trastuzumab-resistant breast cancer, is currently being evaluated in prospective phase III trials for HER2-positive OG cancer in the metastatic setting. Despite improvements in outcome with anti-HER2 therapy, less than half of patients respond to treatment, and predictive biomarkers to lapatinib for breast and OG cancer have not yet been identified.

We sought to identify predictive biomarkers of resistance to lapatinib treatment, common to both breast and OG cancer, by using a siRNA screen of genes identified in a primary siRNA screen to sensitise lapatinib-resistant breast cancer cell lines to lapatinib. These genes are found recurrently amplified and overexpressed in 45 HER2-amplified primary breast cancers and 13 HER2-amplified breast cancer cell lines. OG junction cell lines were treated with lapatinib and screened using the siRNA library. We found that silencing of TP53INP1, a gene target of TP53 which is known to be a reactive oxygen species scavenger, resulted in significant sensitisation to lapatinib in two of the most lapatinib-resistant HER2-positive cell lines derived from the OG junction. TP53INP1 was over-expressed, but not amplified, in the sensitised OG junction cell lines.

Our results suggest that genes which mediate resistance to anti-HER2 therapy in breast cancer may be relevant to OG cancer. We are currently developing assays for TP53INP1 in tumours for validation within the context of prospective randomised controlled clinical trials for OG cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3039. doi:10.1158/1538-7445.AM2011-3039
Abstract 3040: Characterization of a lung cancer growth factor, LASEP1 as a serological biomarker and a therapeutic target
Authors: Takano Atsushi , Daigo Yataro , Nakamura Yusuke
Published: June 2012

Lung cancer is the most common cause of cancer deaths worldwide. To identify molecules that might serve as biomarkers or targets for the development of novel molecular therapies, we have been screening genes encoding transmembrane/secretory proteins that are up-regulated in lung cancers, using cDNA microarrays, and identified a secreted protein, LASEP1 (lung cancer-associated serum protein 1). Immunohistochemical staining using tumor tissue microarrays consisting of 374 non-small cell lung cancers (NSCLCs) confirmed that LASEP1 protein was frequently over-expressed in various histological types of lung cancers; positive staining of LASEP1 was observed in 210 (56.1%) of 374 NSCLCs, while it was observed in 9 (69.2%) of 13 small cell lung cancers (SCLCs). In addition, a high level of LASEP1 expression was associated with poor prognosis for patients with NSCLC (P<0.0001 by log-rank test). Multivariate analysis confirmed its independent prognostic value (P<0.0001 by log-rank test). Serum LASEP1 levels were higher in NSCLC as well as SCLC patients than in healthy volunteers. The proportion of serum LASEP1-positive cases was 127 (38.6%) of 329 lung cancers, while 4 (3.9%) of 102 healthy volunteers were falsely diagnosed. Furthermore, treatment of lung cancer cells with siRNAs against LASEP1 suppressed its expression and resulted in growth suppression of the lung cancer cells, whereas induction of exogenous expression of LASEP1 conferred growth-promoting activity and enhanced the cell mobility in vitro. We found a LASEP1 receptor (LASEPR) which interacts with LASEP1 on lung cancer cell surface. Suppression of LASEPR expression by siRNAs inhibited the growth of cancer cells. LASEP1-LASEPR interaction promoted the cell growth in an anticrime/paracrine manner. LASEP1 is a potential target for the development of diagnostic and prognostic biomarkers as well as therapeutic drugs for lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3040. doi:10.1158/1538-7445.AM2011-3040
Abstract 3042: TPX2 and AURKA promote 20q amplicon driven colorectal adenoma-to-carcinoma progression
Authors: Sillars-Hardebol Anke H. , Carvalho Beatriz , Tijssen Marianne , Beliën Jeroen A.M. , de Wit Meike , Delis-van Diemen Pien M. , Pontén Fredrik , van de Wiel Mark A. , Fijneman Remond J.A. , Meijer Gerrit A.
Published: June 2012

Background: Gain of a large segment of chromosome 20q is associated with progression of colorectal adenomas into carcinomas, implying that multiple genes on the 20q amplicon drive carcinogenesis. Candidate driver genes are expected to be expressed at mRNA and protein levels that correlate with the 20q amplicon DNA copy number status, while functionally affecting one or several cancer-related processes. Integration of CGH profiles with mRNA profiles of a series of colorectal tumors revealed thirty-two candidate genes whose DNA copy number status correlated with mRNA expression levels.

Aim: To functionally analyse the effects of the candidate oncogenes on cancer-related processes by downregulation using siRNA strategies.

Results: Downmodulation of TPX2 (20q11.2) and AURKA (20q13.2) mRNA expression in CRC cell lines with 20q gain affected cell viability, anchorage-independent growth, and invasion. Moreover, immunohistochemical evaluation demonstrated a significant correlation between their protein levels and 20q DNA copy number status in a series of colorectal adenomas and carcinomas.

Conclusion: These data demonstrate that at least two genes located on distinct regions of chromosome 20q promote colorectal adenoma-to-carcinoma progression and indicate that TPX2, like AURKA, is a promising target for anti-cancer drug development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2011-3042
Abstract 3049: Pathways of transformation in Kit-activated melanoma
Authors: Liang Ruixia
Published: June 2012

KIT-activated melanoma is a recently recognized distinct genetic form of melanoma characterized by constitutively activating KIT mutations. To understand the biology of KIT-activated melanoma, we sought to develop both in vitro and in vivo models. Mouse Kit L575P, the orthologue of human KIT L576P, a common KIT mutation found in human melanoma, was expressed in an immortalized but non-transformed mouse Ink4a/Arf-deficient melanocyte cell line. The resultant Kit L575P cell line exhibited increased proliferation, the ability to grow in soft agar and increased migration, indicating that the cells had been transformed by Kit L575P. Furthermore, when Kit L575P cells were injected subcutaneously into NOD/SCID/gamma(c) mice, tumors with all of the histological and clinical hallmarks of melanomas arose in 5 of 7 (71%) mice, while control cells harboring wild-type Kit failed to develop tumors. In addition to a subcutaneous tumor, 1 of 7 mice (14%) injected with Kit L575P cells developed metastatic disease to liver and lungs. Evaluation of signal transduction pathways downstream of constitutively activated Kit L575P revealed striking activation of the phosphatidyl inositol 3-kinase (PI3K) pathway. Inhibition of the PI3K pathway pharmacologically with the PI3K inhibitor LY294002 or genetically by engineering a second Kit mutation (Y719F) which abrogated binding of Kit L575P to the p85α subunit of PI3K, abolished the transformation phenotypes gained in the L575P single mutant. These studies validate this Kit L575P-activated model of melanoma and establish the PI3K pathway as a dominant signaling pathway downstream of Kit in melanoma. This model should be of further use in the pre-clinical evaluation of various therapeutic strategies in Kit-activated melanoma and for studying different aspects of the biology of Kit-activated melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3049. doi:10.1158/1538-7445.AM2011-3049