bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 747: Development and evaluation of rational combination therapy with imidazoacridinones for human bladder cancer patients through the COXEN algorithm
Authors: Smith Steven C. , Havaleshko Dmytro , Moon Kihyuck , Baras Alexander S. , Bekiranov Stefan , Burke Daniel J. , Lee Jae K. , Theodorescu Dan
Published: February 2011

In most patients, bladder cancer metastasis is incurable with current chemotherapy. To try to address this challenge, we have recently employed the novel COXEN algorithm (1) for in silico drug discovery. This approach utilized publicly available gene expression and in vitro sensitivity data for >40k compounds in NCI-60 cell line screen, from which concordant gene expression prediction models from COXEN enabled our discovery of an imidoazacridinone drug, NSC-637993, with high activity in vitro against forty bladder cancer cell lines (2). Examination of additional “hits” provided by COXEN revealed another compound in this class, C1311 (NSC-645809, Symadex) which is undergoing clinical trials in other tumor types.

Our objective was to develop the foundation for use of C1311 in bladder cancer, alone and in rational combination with other clinically relevant agents. Herein, we demonstrate in a panel of 40 human bladder cancer cells that in vitro cytotoxicity profile for C1311 closely correlates that of NSC-637993 and compares favorably to that of standard of chemotherapeutics. Using genome-wide patterns of synthetic lethality of C1311 with ORF knockouts in budding yeast, we determined that combining C1311 with taxanes could provide mechanistically rational combinations for patients that have failed first line therapy with platinum based chemotherapy. To evaluate the biological relevance of these yeast findings in bladder cancer, we evaluated C1311 singly and in doublet combination with paclitaxel in the hollow fiber assay in mice, observing striking efficacy of the agents in vivo. Last, applying COXEN gene expression prediction from 40 bladder cancer cell lines to human patient tumors with known cisplatin-based chemotherapy response data (N=30) (3), we provide evidence that signatures of C1311 sensitivity exist even within patients exhibiting no change or progressive disease on standard of care agents.

Taken together, these findings provide an exemplar for COXEN-based drug discovery and development, while demonstrating the utility of yeast genetics in guidance of rational combinations in vivo. Most importantly, the high activity of these agents in vitro and in vivo calls for clinical trials of these agents, while COXEN provides genomic biomarkers that may be used for patient selection.

(1) Crunkhorn S. Nat Rev Drug Discov 2007;6:782-3.

(2) Lee JK et al. PNAS 2007;104:13086-91.

(3) Als et al. Clin Cancer Res. 2007 Aug 1;13(15 Pt 1):4407-14.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 747.
Abstract 754: Development of death receptor agonists for breast cancer therapy
Authors: Srivastava Rakesh K. , Jackman Chris , Davis Rachel , Shankar Sharmila
Published: February 2011

TRAIL and death receptor (TRAIL-R1 and TRAIL-R2) agonistic antibodies have been shown to induce apoptosis in cancer cells and their clinical trials have produced positive results. However, there are no small organic molecular weight compounds that bind to TRAIL-death receptors and induce apoptosis specifically in cancer cells. The purpose of this study was to identify and characterize death receptor agonists (DRAs)/ small molecular weight organic compounds that induce apoptosis in breast cancer cells. The pharmacokinetics of the DRA with a single oral dose was characterized in nude mice. Blood was collected over 72 hour period after drug administration, and analyzed by HPLC-MS/MS for plasma drug concentration. The Cmax, tmax, and AUC of DRA was measured. DRAs inhibited cell viability and colony formation, and induced apoptosis in estrogen-dependent (MCF-7) and independent (MDA-MB-231 and MDA-MB-468) human breast cancer cell lines, but had no effect on human normal mammary epithelial cells and hepatocytes. The ability of DRAs to induce apoptosis was further enhanced in the presence of TRAIL. DRAs formed active TRAIL-DISC (death-inducing signaling complex) similar to that of TRAIL. DRAs inhibited the growth of orthotopically implanted MDA-MB-231 and MDA-MB-468 tumors in nude mice. Histological examination of tumor tissues derived from DRAs-treated nude mice revealed massive caspase-3 activation and apoptosis. Pharmacokinetic data demonstrate that DRA possesses a long terminal half-life, which is acceptable for further clinical development. In conclusion, we have identified, for the first time, DRAs that can be used in the clinic for the treatment of human breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 754.
Abstract 757: Identification and characterization of small molecule activators of BRCA1 expression
Authors: Harlan-Williams Lisa M. , Overton Jeffrey A. , Stecklein Shane R. , Wang Wenjia , Jensen Roy A.
Published: February 2011

Breast cancer susceptibility gene 1 (BRCA1) has been intensely investigated in normal and cancerous cells and mutations in the BRCA1 gene have been found to account for half of the hereditary breast cancer cases. Although the role of BRCA1 in sporadic breast cancer is still uncertain, 30-40% of such cases show downregulation of BRCA1 expression. Because BRCA1 is a tumor suppressor gene, decreased expression of BRCA1 accelerates growth of mammary tumor cells, while increased expression leads to growth arrest and apoptosis. Furthermore, overexpression of BRCA1 in the murine mammary gland provides protection against mutagen-induced mammary neoplasia. In 2009, the American Cancer Society estimates that 254,650 new cases of invasive and in situ breast cancer will be diagnosed among women. Based on estimates of BRCA1 dysfunction in sporadic breast cancers, over 80,000 newly diagnosed malignancies would have decreased BRCA1 expression. Therefore, a novel prevention or therapeutic strategy targeting BRCA1 could have significant impact on incidence and/or survival.

We developed an MCF-7 breast cancer cell line containing stably integrated copies of a BRCA1 promoter-driven firefly luciferase reporter plasmid and screened over 100,000 compounds for their ability to increase BRCA1-firefly luciferase expression. Six hundred and sixty two compounds (hits) increased BRCA1-firefly luciferase activity greater than 2 standard deviations above the control, DMSO. Secondary dose-response BRCA1-firefly luciferase assays confirmed 27 of the top 32 hits as true active compounds. Potency and efficacy of the active compounds were characterized quantitatively as effective concentration (EC50) and area under the curve (AUC), respectively. Cell viability assays determined that the active compounds had little cytotoxic effects. Non-cytotoxic compounds that increased BRCA1-firefly luciferase activity in a dose-dependent manner with a potency (EC50) and efficacy (AUC) greater than or equal to that of genistein, the positive control, were selected to move forward as lead compounds. Thus far, several lead compounds have been identified. Western blot analysis indicates lead compound 17 increases endogenous BRCA1 expression. Furthermore, DNA foci formation assays indicate compound 17 enhances the ability of BRCA1 to localize to sites of ionizing radiation-induced DNA damage. Because BRCA1 appears to play a role as a growth inhibitor and tumor suppressor in adult breast tissues, identification and characterization of a plausible active compound that enhances BRCA1 expression could potentially translate into a novel prevention or therapeutic option for patients afflicted with breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 757.
Abstract 759: Discovery of MK-8033, a highly specific c-Met/Ron dual inhibitor for the treatment of cancer
Authors: Northrup Alan
Published: February 2011

MK-8033 is a highly specific c-Met/Ron dual inhibitor currently under investigation in Phase I clinical trials for the treatment of cancer. The discovery of MK-8033 is described including the first public disclosure of its structure. Additionally, an X-ray co-crystal structure of MK-8033 with the activated form of c-Met will be discussed to address why, contrary to established dogma, an inhibitor of an activated kinase conformation can lead to high specificity. The in vitro and in vivo properties of MK-8033 as well as aspects of the structure-activity relationships in this class will also be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 759.
Abstract 762: 14-Aminocamptothecin: Synthesis, preclinical activity, and potential use for cancer treatment
Authors: Duan Jian-Xin , Cai Xiaohong , Sun Jessica D. , Meng Fanying , Liu Qian , Bhupathi Deepthi , Ahluwalia Dharmendra , Huang Heli , Jung Don , Hart Charles , Matteucci Mark
Published: February 2011

Topoisomerase I targeted camptothecin analogs are currently employed in a number of different anti-cancer therapeutic regimens. Although efficacious, there continue to be active efforts to develop new camptothecin analogs that avoid deficiencies of the current drugs. The extensive historical SAR efforts on camptothecin and its analogs have largely ignored the 14 position. Only the chloro derivative has been reported, and it is inactive, suggesting a lack of tolerance for substitution at that position. We have developed a highly regioselective process for the synthesis of 14-nitro and 14-aminocamptothecin. 14-aminocamptothecin (TH-1320) demonstrated comparable cytotoxic potency relative to the parent camptothecin, and to clinically relevant camptothecin analogs (topotecan, SN-38) when tested on a panel of human cancer cell lines in vitro (H460, PC3, HT29). Unlike most camptothecin analogs in clinical trials, TH-1320 is not a substrate for any of the major clinically relevant efflux pumps, (MDR1, MRP1, and BCRP). Unlike camptothecin, the potency of TH-1320 is minimally affected by the addition of human serum albumin to cell culture media. TH-1320 showed good bioavailability and brain penetration when dosed orally in mice. TH-1320 is tolerated up to 30 mg/kg with daily oral dosing (MTD). An H460 human NSCLC xenograft model in nude mice was produced by s.c. implantation of 1 ×106 cells into the flank of the animals. When tumor size was approximately 150mm3, animals were randomized into groups of 10 mice each for dosing. 30mg/kg of TH-1320 treatment yielded 79% tumor growth inhibition (TGI) and 15 days tumor growth delay (TGD), while the body weight loss was less than 4%, which suggested a good therapeutic index. Two out of 10 animals showed complete response in the study. However, in same model, Topotecan daily dose at 2mg/kg (MTD) showed only 46% TGI. A xenograft model of metastatic ovarian cancer was set up by the intraperitoneal (i.p.) injection of 3×106 IGROV1 human ovarian cancer cells in nude mice. Mice were evaluated twice a week for morbidity and mortality. Kaplan-Meier survival curves were plotted. Vehicle treated animals had a median survival time (MST) of 39 days. TH-1320 treatment significantly prolonged the survival time. MST of 2/3 MTD dose of TH-1320 (20mg/kg, QDx5/wk x 2wks) was 78 days (p<0.05 vs. vehicle by Logrank test), which was similar to the MTD dose of Topotecan (2mg/kg) or Karenitecin (1.5mg/kg) (MST was 72.5 and 71 days, respectively). Given the favorable properties of TH-1320, further effort is warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 762.
Abstract 764: 6-Bromoindirubin-3′-oxime induces apoptosis of human melanoma cells associated with inhibition of JAK/STAT3 signaling
Authors: Liu Xiangli , Nam Sangkil , Tian Yan , Kritsanida Marina , Magiatis Prokpios , Skaltsounis Leandros , Jove Richard
Published: February 2011

Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively activated in many diverse human tumors and contributes to malignant progression in part by preventing apoptosis. Janus kinases (JAKs), which are receptor-associated tyrosine kinases, phosphorylate STAT3 in normal as well as tumor cells in response to stimulation with cytokines or growth factors. Thus, the JAK/STAT3 signaling pathway has emerged as a promising new target for development of anti-cancer therapeutics. Previous studies demonstrated that bromoindirubin derivatives have anti-cancer activity with enhanced solubility and bioavailability compared to indirubin, which is a natural product with anti-tumor activity found in traditional herbal medicines. The marine natural product derivative 6-bromoindirubin-3′-oxime (6-BIO) has been shown to inhibit kinase activity of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3β (GSK-3β). Here, we report that 6-BIO induces apoptosis of A2058 melanoma cells, associated with inhibition of JAK/STAT3 signaling. Our data show that 6-BIO inhibits viability and induces apoptosis of melanoma cells with an IC50 value of 5 µM, consistent with reduction of phosphorylation of JAK family kinases (JAK1, JAK2 and TYK2) and STAT3 in both dose- and time-dependent manners. Expression of STAT3 downstream targets involved in preventing apoptosis, particularly Mcl-1, was decreased through inactivation of STAT3 by 6-BIO at similar IC50 values. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the AKT and MAPK signaling proteins were not inhibited in the cells treated with 6-BIO. Taken together, these data demonstrate that JAK/STAT3 signaling was selectively inhibited by 6-BIO associated with induction of tumor cell apoptosis. Our findings support further development of 6-BIO as a potential anti-cancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 764.
Abstract 769: Effect of cucurbitacin B, a medicinal herb extract from Trichosanthes cucumerina, on Wnt signaling pathway in breast cancer cells
Authors: Dakeng Sumana , Duangmano Suwit , Jiratchariyakul Weena , Bögler Oliver , Patmasiriwat Pimpicha
Published: February 2011

The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anticancer and anti-inflamatory activities. In this study, we investigated the anticancer activity of cucurbitacin B and dihydrocucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina. A mixture compound of cucurbitacin extracts containing cucurbititacin B and dihydrocucurbitacin B in an 8:1 molar ratio was dissolved in 10% dimethylsulfoxide and diluted with DMEM to the desired concentrations prior to use. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that mixture of cucurbitacin B (mixture CuB) from T. cucumerina have cytotoxic effect on breast cancer cell lines, SKBR-3, MCF-7, HBL-100 and T47D with the IC50 of 4.60 µg/ml, 88.75 µg/ml, 55.46 µg/ml and >100 µg/ml respectively. Flow cytometry for cell cycle analysis was performed after treated cells with mixture CuB. We found that growth inhibition effect was attributed at G2/M phase arrest and apoptosis. Western blotting analysis showed increase PARP cleavage and decrease Wnt associated signaling molecules including β-catenin, galectin-3, cyclin D1 and c-Myc. We also found that mixture CuB inhibit nuclear-translocation of β-catenin and galectin-3 when compared with vehicle control cells. The depletion of β-catenin and galectin-3 in the nuclease was confirmed by cellular protein fractionation method. TCF promoter, a downstream signaling of Wnt was also disrupted in cucurbitacin B treated cells. TCF promoter assay system was used for assessment of TCF promoter activity. The relative luciferase activity was reduced when we treated cells with mixture CuB for 24 hours. Our data indicate that cucurbitacin B and their derivative dihydrocucurbitacin B from T. cucumerina induce apoptotic cell death and exhibit growth inhibitory effect via inhibition of Wnt signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 769.
Abstract 78: Role of 12-lipoxygenase in regulation of ovarian cancer cell proliferation and survival
Authors: guo austin M. , Liu Xiuli , Al-Walhab Zaid , Maddippati Rao , Scicli A. Guillermo , Munkarah Adnan
Published: February 2011

Enzymes involved in eicosanoid synthesis have previously been implicated in the progression of various types of cancer, including ovarian carcinomas. Most of the current data in ovarian cancer have focused on cyclooxygenase-2 (COX-2). It is not known whether the main human 12-hydroxyeicosatetrenoate (12-HETE) generating enzyme, 12-lipoxygenase (12-LOX), contributes to the regulation of growth of ovarian cancer cells. In the present study, we examined the function of the 12-lipoxygenase pathway in the regulation of proliferation and survival of three epithelial ovarian cancer (EOC) cell lines. We found that all three EOC cells express high level of the mRNA encoding for 12-LOX, using RT-PCR. These cells also contain significant amount of immuno-reactive 12-LOX protein as revealed by western blot. Liquid chromatography/Mass Spectrometry (LC/MS) analysis confirmed that all three EOC produce 12-HETE when challenged with arachidonic acid (AA). N-benzyl-n-hydroxy-5-phenyl-pentanamide (BMD-122) is a specific 12-LOX inhibitor. Exposure of the three EOC cell lines to an increasing dose of the 12-LOX inhibitor, BMD-122 (1 μM, 10 μM and 50 μM), resulted in a dose-dependent inhibition in cell proliferation (75±9%, 51±6% and 26±4% of untreated control, respectively), measured by MTT assay in vitro. Furthermore, treatment with 10 μM BMD-122 was associated with up to ∼30% increases in the apoptosis of these EOC cells within 24 hr, measured by Annexin-V flow cytometry. This apoptosis seems to be mediated through a mechanism independent of PI-3 kinase since the expression of phosphorylated Akt was not affected by BMD-122 treatment. Furthermore, we found that BMD-122 treatment had no effect on the three EOC cell lines ability to invasion using Matrigel assay. Taken together, these data suggest that 12-LOX is involved in regulation of ovarian cancer progression and survival, an important and novel finding. Inhibition of 12-LOX may have potential as an adjuvant therapy in ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 78.
Abstract 781: Bioconversion of curcumin and its analog by Beauveria bassiana ATCC 7159
Authors: Martin Glenroy , Ebert Tamara , McPhee Jennifer , Mercer Bettie , Williams Jesica
Published: February 2011

Curcumin (1), a chemopreventive agent from turmeric (Curcuma longa L.) was incubated with the microbial cultures of Beauveria bassiana ATCC 7159. Bioconversion of [(1E, 6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione] (1) with B. bassiana afforded five major metabolites: 1,7-bis(4-hydroxyphenyl)heptane-3,5-dione (2), 3-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hept-1-ene (3), 1,2,6,7-diepoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-3,5-diol (4), 5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hept-1-ene (5) and 1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3,5-diol (6). The biotransformation of the synthetic analog 1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (7) by B. bassiana afforded the reduced diol 1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3,5-diol (6) as the only metabolite.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 781.
Abstract 782: Characterization of genetic changes in oligodendroglial tumors including glioblastomas with oligodendroglial component
Authors: Klink Barbara , Krex Dietmar , Gerlach Eva-Maria , Schlingelhof Ben , Hackmann Karl , Patt Stephan , Schrock Evelin
Published: February 2011

In contrast to astrocytic tumors including glioblastomas (GBM), oligodendrogliomas (O) show a better prognosis and increased responsiveness to chemotherapy. Interestingly, a small subgroup of glioblastomas contains areas with histological features of oligodendroglial differentiation. In the current WHO classification (2007) these tumors are now included as a variant of glioblastomas and are called glioblastomas with oligodendroglial component (GBMO). However, definitive diagnostic criteria still do not exist.

In this study we used a genome wide approach (chromosomal comparative genomic hybridization (CGH) and molecular karyotyping) in combination with interphase fluoreszence in situ hybridization (FISH) (regions 1p, 1q, 7q, 10q, 17p, 19q, cen18, 21q) to genetically characterize GBMO and “classical” O. We analyzed the oligodendroglial and the astrocytic glioblastoma parts of 13 GBMO separately by chromosomal CGH of microdissected paraffin embedded tumor tissue and interphase FISH on paraffin sections. Furthermore, freshly frozen material of six oligodendrogliomas (4 WHO grade II, 6 WHO-grade III) was examined genome wide by using the standard Agilent 244A chip.

We identified four distinct genetic subtypes in GBMO: an “astrocytic” subtype (9/13) characterized by +7/-10; an “oligodendroglial” subtype with −1p/-19q (1/13); an “intermediate” subtype showing +7/-1p (1/13), and an “other” subtype having none of the former aberrations typical for gliomas (2/13). In contrast, most “classical” O (5/6) showed combined 1p/19q deletion, corresponding genetically to the “oligodendroglial” subtype. Four of the O with 1p/19q codeletion showed additional aberrations (e. g. −4, −14, −9, −18). The different histological tumor parts of GBMO revealed common genetic changes in all tumors and showed additional aberrations specific for their oligodendroglial and astrocytic parts. These findings demonstrate the monoclonal origin of the tumor followed by the formation of the astrocytic and oligodendroglial components. We are currently evaluating the array CGH results of six oligodendrogliomas, which will also be presented.

The diagnostic determination of these genetic signatures may allow for a better prognostication of the patients. Our results underline the importance of molecular cytogenetic analyses to supplement the histological diagnosis of gliomas.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 782.
Abstract 784: Plasmacytoid urothelial carcinoma: Molecular and histopathological characterization of a rare variant of bladder cancer
Authors: Stoehr Robert , Keck Bastian , Rogler Anja , Hofstaedter Ferdinand , Fritsche Hans-Martin , Lehmann Jan , Montironi Rodolfo , Sibony Mathilde , Wullich Bernd , Hartmann Arndt
Published: February 2011

AIM: The plasmaycytoid urothelial carcinoma (PUC) is a rare variant of urothelial carcinoma. To date only few cases have been reported and molecular data are not published yet. Therefore, we analysed clinical, histopathological and molecular features in a series of 32 PUC.

METHODS: Expression of CK7, CK20, PAN-CK, p53, Ki67, CD138, E-cadherin and beta-catenin was analysed immunohistochemically on a tissue microarray. UroVysion FISH was performed to asses ploidy status. After microdissection and DNA isolation, FGFR3 and PIK3CA mutation status were determined using SNaPshot analysis. P53 mutation analysis was performed by direct sequencing of exons 5-9. Clinical data available from 16 PUC were compared to 269 patients with conventional locally advanced urothelial bladder cancer within a randomized multicenter trial (AUO-AB 05/95).

RESULTS: PUC were usually diagnosed in advanced pathologic stage (64% pT3, 23% pT4) with 60% metastatic disease. Overall survival of PUC treated with cystectomy and adjuvant chemotherapy was lower than of patients with conventional locally advanced bladder cancer (23.3 vs. 49.9 months, p=0.051). 87% of PUCs showed loss or strongly reduced expression of E-cadherin. Beta-catenin was completely absent in 22.5% and associated with loss of E-cadherin (p=0.033). 16.7% of PUC showed a nuclear staining for beta-catenin, which was more frequent in E-cadherin negative PUC. CK20 expression was abnormal in all cases. 22.6% of PUCs were negative for CK7. 97% of PUC displayed a positive PAN-CK staining. 78% coexpressed CD138. Nuclear accumulation of P53 was found in 68.4% of E-cadherin negative PUC, but only in 27.2% of E-cadherin positive ones. p53 mutations were found in 9/31 cases. 69% of PUC showed increased Ki67 labelling index (>15%). FISH displayed PUC as highly aneuploid tumors. Relative deletion of 9p21 was found in 70%. No mutations were found in FGFR3 and PIK3CA.

CCONCLUSIONS: PUC showed molecular and histopathologic features of aggressive bladder cancer. Prognosis in locally advanced PUC treated with adjuvant chemotherapy seems to be unfavourable in comparison to conventional bladder cancer. Loss of E-Cadherin is present in the majority of PUC and is associated with a higher degree of p53 and beta-catenin accumulation, which is in line with the high metastatic potential of PUC. CD 138 is not suitable for differential diagnosis to plasmocytoma. PAN CK is an important diagnostic tool for clarification of the epithelial origin of PUC as there are cases negative for CK7 and CK20.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 784.
Abstract 789: Determination of MAPK and Wnt pathway activity in esophageal cancer
Authors: Shannon Nicholas B. , Peters Christopher J. , Rees Jonathan R. , Hardwick James S. , Zhang Chunsheng , Hardwick Richard H. , Fitzgerald Rebecca C.
Published: February 2011

Introduction:

Gastro-esophageal adenocarcinomas are rapidly increasing in incidence and have a five year survival of less than 14%. Analysis of signaling pathways in gastro-esophageal adenocarcinomas can be used as a route to identifying possible therapeutic targets.

Aims and methods:

We had already identified MAPK and Wnt pathways as important in this disease. The aim of this work was to identify biomarkers of pathway activation for future application to primary tumors for selection of targeted therapies.

Three cell lines (CP-B, CP-C, CP-D) without constitutive activation were stimulated with exogenous ligands for the Wnt or MAPK or pathways. The TGFβ pathway was also stimulated as a negative control to test biomarker specificity. The expression of potential biomarkers of transcriptional activity was then profiled using real time PCR. To determine applicability of these markers to human tissue the expression of these markers was assessed in an expression array data set consisting of 75 gastro-esophageal adenocarcinomas.

Results:

Out of 8 (MAPK) and 13 (Wnt) potential markers, three markers were selected as specific for the MAPK pathway (EGR-1, JUN and FOSL1) or the Wnt pathway (BMP4, TCF7, LEF1). All markers had upregulation of expression of at least 2 fold that were specific to activation of the relevant pathway (Table 1).Table 1.Fold changes of expression upon activation by stimulation with exogenous ligandsMarkerEGF (MAPK pathway)LiCl (Wnt pathway)TGFβ (TGFβ pathway)P-value (ANOVA)EGR-1130.50.91.3< 10-4JUN3.31.21.0< 10-4FOSL111.02.60.6< 10-4BMP42.18.31.5< 10-4LEF10.523.30.5< 10-4TCF72.44.80.7< 10-4

Next we assessed the expression of selected markers in 75 gastro-esophageal adenocarcinomas for which expression profiling data was available. In these tumors 27 (36%) had activation of the MAPK pathway alone, 10 (13%) had activation of the Wnt pathway alone, and a further 12 (16%) had activation of both pathways.

Conclusions:

We have determined a set of markers which can be used to assess pathway activation of the MAPK and Wnt pathways in gastro-esophageal adenocarcinomas. External validation of these pathway activation markers is ongoing using multiplex PCR as a prelude to a clinical trial of targeted therapy.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 789.
Abstract 79: The compartmentalization of folate metabolism depends on two isoforms of folylpoly-γ-glutamate synthetase and the mitochondrial folate transporter
Authors: Lawrence Scott A. , Moran Richard G.
Published: February 2011

The reactions of folate metabolism are distributed between the cytosolic and mitochondrial compartments of mammalian cells and both components are essential for mammalian cell survival. The enzyme folylpoly-γ-glutamate synthetase (FPGS) has been shown to be essential for the intracellular retention of folates as polyglutamate derivatives. The fpgs gene produces two transcripts, one for an isoform with a leader peptide targeting it to the mitochondrial matrix, and the other is transcribed without this sequence, so that the encoded isoform remains in the cytosol. Transfer of folates into the mitochondrial matrix is mediated by an inner mitochondrial membrane transport protein, the mitochondrial folate transporter (MFT). Either deletion of the MFT or of mitochondrial FPGS is cytotoxic and renders the cell auxotrophic for glycine; the mechanism of this effect is far from apparent. To better understand this compartmentalization, we generated cell lines that express only the cytosolic or mitochondrial isoform of FPGS under tetracycline-inducible control in FPGS-null AuxB1 cells. Western blot analysis of the FPGS protein in these cell lines indicate that both FPGS isoforms are expressed near endogenous levels in the presence of doxycycline and that each cell line solely expresses the FPGS isoform in the respective compartment. Clonogenic survival of each doxycycline-induced cell line demonstrates a cellular requirement for each FPGS isoform. Additional studies showed that cells complemented with cytosolic FPGS only accumulated and retained folates in the cytosol but not mitochondria, leading us to conclude that folate polyglutamates are not substrates for the MFT. In addition, expression of only the mitochondrial form of FPGS resulted in cells that progressively trapped folates in the mitochondria, leading us to the concept that folate polyglutamates do not escape the mitochondria. However, cytosolic folates were observed in cells expressing only mitochondrial FPGS. We are currently using these cell lines to study the transfer of folates across the mitochondrial membrane and polyglutamation in each compartment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 79.
Abstract 796: Molecular characterization of circulating tumor cells using fluidigm biomark dynamic array
Authors: Jiaqi Huang , Brohawn Philip , Bao Haifeng , Burke Patricia , Chen Xiaoru , Zhu Wei , LaVallee Theresa , Jallal Bahija , Yao Yihong
Published: February 2011

Circulating tumor cells (CTCs) are cancer cells derived from a primary tumor and/or its metastases that circulate in the peripheral blood. The number of CTCs in whole blood of cancer patients has been shown to have clinical relevance with respect to patient prognosis. There is, in addition to CTC enumeration, great interest in the molecular characterization of isolated CTCs. However, there are challenges for molecular characterization of CTCs with commonly used methodologies. The number of CTCs is extremely low and despite the depletion of leukocytes during cell enrichment, the CTC-enriched fractions still include numerous leukocytes which strongly impact CTC-specific gene expression profiling. CellSearch™ CTC test is a FDA approved diagnostic test for enumeration of CTCs. The fixation reagents in the CellSave tube allow processing of samples for several days post-collection, making analysis of clinical samples more practical compared to near-same day processing required for samples collected in EDTA tubes. However, the fixation decreases the mRNA signal and assay sensitivity. We have established a protocol which overcomes these hurdles and allows the profiling of select transcripts of interest in CTCs. By applying a proteinase K treatment and Trizol LS lysis prior to RNA isolation, we were able to overcome the impact of fixation on RNA isolation. RNA was isolated from the treated lysate using a commercially available kit applicable to low cell numbers. Use of this kit allowed us to utilize all of the isolated RNA in the cDNA step. Superscript III cDNA synthesis was then carried out according to the manufacturer's protocol. Target genes of interest were then pre-amplified using the Life Technologies established methodology and reagents. The resulting volume of pre-amplified cDNA was concentrated to a set volume to maximize the amount of transcript profiled. Genes of interest were profiled on the Fluidigm Biomark 48.48 Array. mRNA expression analysis was performed on genes which are not expressed in a pure leukocyte population to ensure that any signal detected was from the CTCs, and not from the background leukocyte population. Using this methodology, we succeeded in performing quantitative gene expression analysis on genes such as PDGFRα on as few as five tumors cells (MG63) fixed in CellSave tubes and spiked into a background environment of contaminating leukocytes (typically 1000 or more) pulled down from normal blood using the CellSearch system. These methods could potentially be used not only for greater understanding of the biology and metastatic potentialof CTCs

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 796.
Abstract 800: Novel GMDS mutation and clinical estimation of fucosylation in several kinds of human cancer
Authors: Moriwaki Kenta , Tsuda Saori , Shinzaki Shinichiro , Furukawa Yoichi , Miyoshi Eiji
Published: February 2011

Oligosaccharides are some of the most important factors in the posttranslational modification of proteins and lipids. It is well known that oligosaccharide structures change during malignant transformation. Fucose is a type of constituent of oligosaccharides, and is notably associated with cancer and inflammation. Fucosylation is catalyzed by several kinds of fucosyltransferases, the GDP-fucose synthetic enzymes, and GDP-fucose transporter. Recently, we found a mutation of the GMDS gene, which involved in the synthesis of GDP-fucose, in a colon cancer cell line, HCT116, leading to a virtually complete deficiency of fucosylation. Down-regulation of cellular fucosylation rendered the cells resistant to tumor necrosis factor-related apotosis-inducing ligand (TRAIL) -induced apoptosis, resulting in the escape from natural killer (NK) cell-mediated tumor immune surveillance. Thus, GMDS and fucosylation are thought to be regulatory factors for TRAIL-induced apoptosis and tumor immune surveillance.

In the present study, we searched mutation of the GMDS gene in human cancer cell lines and various kinds of cancer tissues. The aberrant transcripts with deletions of exons 2-4 and 5-7 were detected in the cDNA of LS174T and NCI-H716 cells, respectively. Since these mutations were heterozygous, the deficiency of fucosylation was not observed. In a gastric choriocarcinoma cell line, SCH, only short transcript with deletion of exons 2-4 was detected. This cell line showed a marked decrease in the fucosylation level. PCR and subsequent direct sequencing analyses using 100 human colon cancer tissues identified mutations in two cases. One of the two was c.739 C>T (p.R247X) in exon7, and the other was c.345+5 G>A (IVS4+5) in intron 4. Furthermore, RT-PCR analysis identified aberrant transcripts in 5 of 10 ovarian cancer tissues. All mutant GMDS genes found in this study had no enzymatic activity.

Subsequently, the level of fucosylation in many colon cancer tissues was investigated by immunohistochemical analysis using two types of fucosylated oligosaccharide-specific lectin. The decrease of fucosylation was observed in certain cases. Especially, core-fucosylation was decreased in colon cancer with lymph node metastasis.

These results suggest that cancer progression in certain patients with colon cancer is dependent on de-fucosylation. Thus, fucosylation is an attractive target for cancer diagnosis and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 800.
Abstract 801: Clinical application of genomic signature including ALK mutation to the new tumor risk classification for neuroblastoma
Authors: (-Ichikawa) Miki Ohira , Kojima Toshio , Niwa Takafumi , Oba Shigeyuki , Ishii Shin , Takita Junko , Kato Motohiro , Ogawa Seishi , Nakamura Yohko , Kamijo Takehiko , Nakagawara Akira
Published: February 2011

Neuroblastoma (NBL) is one of the most common pediatric tumor arising from developing sympathetic nervous system. Its clinical and biological behavior is markedly heterogenous ranging from aggressive progression despite of intensive therapy to spontanous regression. NBL DNA shows complex patterns of genomic aberrations including MYCN amplification, deletion of chromosome 1p or 11q, and gain of chromosome 17q. We previously constructed the genomic subgrouping system of NBL based on the array CGH analysis for new tumor risk classification of NBL (Tomioka et al, Oncogene 2008). Analysis of 234 tumors by using 2,464 clones-BAC arrays demonstrated there exist three genomic groups (GG) of chromosomal aberrations in NBL: silent (S), partial gains and/or losses (P) and whole gains and/or losses (W). Each GG was further segregated into subgroups with different clinical outcomes clearly defined by MYCN amplification (MYCN-amp), 1p loss, 11q loss and 17q gain. Importantly, high-resolution array CGH analysis also led us to identify a novel genomic alteration in NBL, the activating mutation and amplification of anaplastic lymphoma kinase (ALK) oncogene on chromosome 2p23. In this study, to conduct the clinical validation of our GG risk classification system, array CGH and mutation analysis of ALK gene of 343 sporadic NBLs (stage 1: 48; stage2: 29; stage 4s: 27; stage 3: 60; stage 4: 179; median follow-up period is 78 months) were performed and the clinical impact of the genomic signatures was assessed. The results were fairly reproducible with our previous data, showing that GG-S or GG-W without MYCN-amp exhibited good prognosis (Ss and Ws, 8-year survival rates: >80%), while GG-P or GG-S accompanying MYCN-amp were very poor (Pa and Sa, 8-year survival rates: <35%). ALK gene abnormalities including mutations and amplification were found in 21 tumors in our dataset (6.1%), which caused fatal outcomes of the patients (p<0.01) especially those with MYCN-amp (ALKmut-/MYCNamp+, n=73 vs. ALKmut+/MYCNamp+, n=10, p=0.16). Interestingly, ALK abnormalities were frequently found in the certain genomic subgroups of the tumors, namely, GG-Pa with 1p loss, GG-Ps with 11q loss and GG-Ws with no partial 1p and 11q losses. These results suggested that the combination of genomic signatures with ALK abnormalities will further improve the potential of our tumor risk classification system of NBL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 801.
Abstract 802: Minimal deleted regions and their clinical significances in oral squamous cell cacinomas
Authors: Hsieh Ling-Ling , Chien Huei-Tzu , Huang Shiang-Fu , Chen I-How , Liao Chun-Ta , Wang Hung-Ming
Published: February 2011

With improvements in technologies, DNA alterations on a genome-wide scale have been successfully evaluated in several human cancers. To determine frequently novel deletion regions in oral squamous cell carcinomas (OSCCs) in Taiwan and regions specifically associated with clinicopathologic parameters and prognoses, the 400 polymorphic microsatellite markers from Applied Biosystems PRISM Linkage Mapping Set-MD10 (PE Biosystems, Foster City, CA) were used to examine 63 male primary tumors. To make sure that tumor tissue samples for genome-wide LOH screening consisted >90% tumor cells, LCM was performed on immunostaining slide using a PixCell laser capture microscope (Arcturus Engineering, Mountain View, CA). For increasing reliability of statistical analysis, only when a microsatellite marker was able to provide LOH information in more than 30% (> 19 cases) of the DNA samples was considered to be a successful allelotyping. Based on this criterion, 276 markers were included for the present study. The mean heterozygosity rate in these 276 microsatellite markers was 70.04%. The percentage of LOH among the markers was ranged from 3.85% (marker D20S171) to 65.52% (marker D18S478) with a mean of 33.63% (SD, 12.28%). Twenty-four markers displayed >50% LOH and chromosome regions at 2p21, 2p11.2, 5p15.1, 5q35.1 and 7q21.13 were novel frequent LOH regions not reported previously in OSCC. In this study, we found that markers with a LOH frequency >35% are likely to show LOHs associated with cancer-specific phenotypes. Therefore, only markers with a LOH frequency >35% (121 markers) were used to examine whether there were associations between LOH and the clinicopathologic parameters, including age, anatomic site, tumor stage and risk factors of oral cancer (cigarette smoking, alcohol drinking and AQ chewing) and construct minimal deleted region (MDR). Thirty-two MDRs were defined and 10 novel MDRs were noted. A loss of MDR c19r2 (19q13.42-19q13.43) was correlated significantly with cigarette smoking and a loss of MDR c14r1 (14q24.2-14q32.12) and c11r1 (11q13.4-11q25) was associated with disease-free survival. A loss of MDR c14r1 and c11r1 was associated with the magnitude of genomic instability estimated by the proportion of loci showing LOH (PLOH) and the degree of chromosomal derangement estimated by the mean fractional allelic loss (FAL) in a tumor. In conclusion, our comprehensive allelotype analyses have unveiled a few MDRs involved in the development of OSCC in Taiwan. These results provide useful clues for identification of putative tumor suppressor genes by fine mapping of the suspected MDRs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 802.
Abstract 81: Differential metabolic and viability responses of the A549 and MRC-5 cell lines upon exposure to selective organic inhibitors of glycolysis
Authors: Farah Ibrahim O. , Lewis Veshell L.
Published: February 2011

Lung cancer is a one of the most prevalent and deadly cancers in United States. Research has shown that cancer cells exhibit higher glycolytic rates than normal cells. Studies on the differential role of the interference with glycolysis/energy generation by cancer cells through the use natural glycolytic inhibitors were limited in the literature. Therefore, we hypothesize that exposure of lung cancer cells to natural inhibitors of glycolysis will negatively influence their metabolic activities as well as their cell viabilities due to the inhibition of substrate-level ATP generation, while differentially not affecting the normal lung phenotype. The human lung fibroblast cell line (MRC-5) was selected to represent the normal human lung and the human alveolar epithelial cell line A549 was selected to represent lung cancer in vitro. These cells were maintained and exposed to eleven different organic reagents including fructose diphosphate (FDP), sodium citrate, ascorbic acid, crude honey, sodium bicarbonate, D-glucose, oxalic acid, glycerol, zinc acetate, pyruvic acid, and sodium ascorbate at concentration levels ranging from 31.3-2,000 ug/ml for 24-72 hours in 5% CO2 and 96 well plates using MTT, Alamar blue and the T4 cellometer assays as well as phase-contrast photo-imaging. Our results indicate that exposure of A549 cells to these organics resulted in concentration dependent differential cell destruction of the A549 cell line. Nine of the eleven organics used showed statistically significant (p<0.05) differential negative effects (impaired metabolic activity and cell death) on the A549 cell line in comparison to its control as well as to their effects on the MRC-5 cell line using MTT, Alamar blue and T4 cell viability assays. Data from the MTT assay showed limitations in detecting the true cell viability in comparison to the T4 data. Viability using the T4 cellometer ranged between 60-76% for the A549 compared to 96-100% for its control as well as the exposed MRC-5 cell line. LC50 using MTT and Alamar blue assays ranged between 161-1041 ug/ml. None of the eleven chemicals used allowed for cell proliferation of the A549 cell line while showing no or minor effects on the MRC-5 cell line. We conclude that nine of the tested organics impaired glycolysis, which is crucial to the generation of cellular energy and survival of the A549 cell line. Supported by NIH-RCMI grant# G12RR13459 and NIH-RISE grant # 63228.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 81.