bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 164: High-throughput epigenetic analysis of susceptibility loci identified by GWAS in pancreatic cancer
Authors: Lo Ken C. , Holster Heather , Jia Jinping , Parikh Hemang , Collins Irene , Brazas Robert , Selzer Rebecca , Amundadottir Laufey
Published: February 2011

Pancreatic cancer is a highly lethal cancer with few well established risk factors. A genome wide association study (GWAS) of pancreatic cancer (PanScan) is being conducted within the framework of the NCI-sponsored Cohort Consortium and the Pancreatic Cancer Case-Control Consortium (PANC4). Susceptibility loci discovered to date in PanScan are non-coding variants that lie in intronic or intergenic regions, suggesting that the underlying signals may function through regulatory mechanisms that influence gene expression or splicing. Alternatively, these may lie in unannotated transcripts and directly affect their function.

Epigenetic mechanisms, such as DNA methylation, can affect the regulation of gene expression and plays a critical role in the development of many human diseases including cancer. Powerful methods exist to analyze DNA methylation patterns in higher eukaryotes including methylated DNA immunoprecipitation (MeDIP), an affinity based approach to enrich methylated DNA regions from genomic DNA, which can be combined with microarrays to profile genomic DNA methylation patterns. We created a new semi-custom MeDIP-optimized array design based on our Human DNA Methylation 3×720K CpG Island Plus RefSeq Promoter array by tiling additional regions associated with pancreatic cancer that were identified in the PanScan study. To complement DNA methylation data a genome-wide transcriptome analysis was performed with RNA-sequencing (RNA-seq). Here we describe a comprehensive genome wide analysis of 8 pancreatic cancer cell lines to examine methylation patterns of Refseq promoters and annotated CpG islands as well as transcribed sequences.

One of the susceptibility loci from PanScan is in the vicinity of the ABO gene on Chr9q34 where four SNPs (rs505922, rs495828, rs657152 and rs630014) are associated with a significantly increased risk of pancreatic cancer. As a pilot study, DNA methylation patterns and expressed sequences in this locus were investigated in cell lines derived from pancreatic tumors and normal pancreatic tissues. Our genome wide DNA methylation and RNA-seq analysis aims at establishing a comprehensive catalog of epigenetic patterns in pancreatic cell lines that could provide plausibility for the association signal in the ABO gene and other GWAS regions and thereby, initiate the characterization of the molecular phenotype of the susceptibility alleles for pancreatic cancer risk.

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 164.
Abstract 1642: Preclinical studies of MK-8745: Correlation between Aurora-A inhibitor sensitivity and Aurora-A activator
Authors: Chowdhury Aparajita , Tsai Ming Y.
Published: February 2011

Selective small-molecule kinase inhibitors have emerged as an important class of anti-cancer agents, and have demonstrated encouraging clinical efficacy in several different malignancies. However, it has become clear that the clinical activity of these agents is typically limited to a subset of patients, indicating the need to develop a clear understanding of the various factors that influence clinical benefit. To date, most studies have identified cancer patients whose tumors have genetic alterations of the targeted kinase and have demonstrated their greatest clinical efficacy for the kinase inhibitors. However, recent drug discovery efforts are also being aimed at targeting kinases within oncogene-associated signaling pathways and the pathway-specific determinants of sensitivity. Aurora-kinase family members (Aurora kinases -A, B, C) have been implicated as key mitotic regulators essential for genomic stability. Aurora kinase A (AURKA) is highly expressed in multiple human cancers and has been shown to be a promising therapeutic target. In this study, we examined the effects of small molecule AURKA specific inhibitor MK-8745 on non-Hodgkin lymphoma (NHL) cells.

Our results indicate that MK-8745 treatment leads to cell cycle arrest at G2/M phase with accumulation of tetraploid DNA followed by mitotic catastrophe and cell death in five cell lines (Granta 519, Jeko1, JVM2, Z138C and Akata). More importantly, we found that the sensitivity of the cell to the AURKA inhibitor is strongly correlated to the expression level of AURKA activators. We found that siRNA knockdown of one of the AURKA activators TPX2 increased MK-8745 sensitivity in drug resistant cell lines (Granta 519), whereas overexpression of TPX2 in MK-8745 sensitive cell (Z138C) lines increased drug resistance. This differential drug-sensitivity is specific to MK-8745 since treatment with pan-Aurora inhibitor (VE465, inhibitor of Aurora-A, B and C) and another mitotic drug, Taxol, did not show a differential sensitivity in these cell lines. The AURKA phosphorylation was decreased by MK-8745 treatment with simultaneous reduction in endogenous AURKA substrate TPX2, Eg5 and TACC3 protein levels.

Our results suggest, beside p53, AURKA activators should be potential biomarkers for selecting subpopulations of patients for AURKA inhibitor treatment, while AURKA substrates can be the potential biomarkers for the effect of Aurora-A inhibitor treatment.

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 1642.
Abstract 1643: Withaferin A analogues up-regulate the MAPK and glutathione metabolic pathways prior to activation of apoptosis in pancreatic cancer cells
Authors: Mahadevan Daruka , Liu Xiaobing , Qi Wenqing , Cooke Laurence , Wijevatne Kithsiri , Marron Marilyn , Gunatilaka Leslie
Published: February 2011

Withaferin A is a natural product known to possess anti-cancer activity. Here we studied the effect of Withaferin A and 11 of its analogs on differentiation state-specific pancreatic cancer cell lines. Three pancreatic cell lines Panc-1 (poorly-differentiated), MiaPaca-2 (moderately differentiated) and BxPC-3 (well differentiated) were treated with 11 analogs and cell proliferation was examined by MTS assays. The IC50 of these 11 analogs were <4.0 µM suggesting they have growth inhibitory effects on pancreatic cancer cell proliferation. Two of these 11 analogs, named KW-120-94-1 and KW-120-95-1 were selected for further study. The IC50s of KW-120-94-1 and KW-120-95-1 were 2.25, 0.56 µM (Panc-1); 1.53, 0.52 µM (MiaPaca-2) and 0.45, 0.29 µM (BxPC-3) respectively. Apoptosis was analyzed by morphology (Acridine Orange and Ethidium Bromide staining), flow cytometry (Annexin V-FITC and Propidium Iodide staining) and PARP cleavage. The data showed that apoptosis was induced both in BXPC-3 and MiaPaca-2 cells in a dose-dependent manner. However, Panc-1 cells were in general resistant to apoptosis. In BxPC-3 cells apoptosis was induced as early as 6 hours after KW-120-95-1 treatment at 1.5µM. To determine the Withaferin A analog dependent ‘tumor profile signature’, gene expression profiling by Affymetrix microarrays were performed in the 3 pancreas cancer cell lines. The cells were treated with KW-120-95-1 for 8 hr at 1.5µM. Differential gene expression was analyzed by ANOVA which identified 33 (MiaPaca-2), 54 (Panc-1) and 48 (BXPC-3) gene changes. The Fisher exact test demonstrated that MAPK signaling and glutathione metabolism pathways were over-expressed within the genes that changed with treatment. Western blotting and real-time RT-PCR were utilized to confirm gene expression changes identified in the MAPK pathway (HSPA1A/HSPA1B, c-Jun) and glutathione metabolism pathway (GCLC, GPX2 and GPX3). Taken together, our data indicate that Withaferin A analogs inhibit pancreatic cancer proliferation and induces apoptosis by activating the MAPK and glutathione (stress) pathways. The molecular target(s) of Withaferin A and analogs are likely to function by activating the MAPK signaling and glutathione metabolism pathways.

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 1643.
Abstract 165: MIRA (methylated CpG island recovery assay) assay identifies frequently methylated genes in breast cancer
Authors: Hill Victoria , Hesson Luke , Dansranjavin Temuujin , Dallol Ashraf , Bieche Ivan , Tommasi Stella , Dobbins Timothy , Gentle Dean , Euhus David , Dammann Reinhard , Ward Robyn L. , Minna John , Maher Eammon R. , Pfeifer Gerd , Latif Farida
Published: February 2011

There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of combined bisulphite restriction analysis (CoBRA) and sequencing of bisulfite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumors; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumors, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal and prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly, methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic 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 165.
Abstract 1650: Targeting of compensatory signaling pathways identified in a synthetic lethal screen leads to synergistic growth inhibition in bladder cancer cells
Authors: Axelrod Mark J. , Gioeli Daniel , Weber Michael J.
Published: February 2011

Despite promising preclinical results, monotherapies that target deregulated signal transduction pathways often are insufficient to slow or shrink tumors in human patients. We hypothesize that multiple pathways within the cellular signaling network are altered in cancer cells, and that these pathways are acting in a redundant or compensatory manner to provide resistance against drugs targeting a single protein. A treatment plan consisting of multiple small molecules that inhibit these compensatory and redundant pathways should overcome this resistance. To discover pathways that act in a redundant or compensatory manner, we performed a screen that allows us to examine the cellular effects of blocking pathways both individually and in combination. We found that the combination of the epidermal growth factor receptor (EGFR) inhibitor lapatinib in combination with the phosphoinositol-3-kinase (PI3K) inhibitor LY294002 produced synergistic growth inhibition in UMUC-6 bladder cancer cells. In order to confirm that this effect was due to inhibition of the putative targets of these compounds, we substituted structurally distinct EGFR and PI3K inhibitors for lapatinib and LY294002 and obtained similar results. Cell cycle analysis of cells treated with this combination revealed an arrest in the G1 phase, which could explain the synergistic inhibition observed in the screen. In order to find a molecular mechanism for this synergistic effect, we performed Western blotting analysis of the cells treated with each drug individually and in combination. We found that treatment with LY294002 caused an increase in levels of phosphorylated SHC, and ERK, indicating that receptor tyrosine kinase signaling, and possibly EGFR signaling is activated to compensate for PI3K pathway inhibition. Inhibition of ERK activity with PD325901 produced the same effect as EGFR inhibition, confirming the role of ERK as a compensatory signaling molecule for PI3K inhibition. Parallel RNAi experiments against SHC are currently ongoing. These data suggest that synthetic lethal screening with a library of targeted inhibitors of signaling proteins can be used to search for combinatorial effects and functionally identify compensatory and redundant relationships between different signaling components. This work paves the way for the development of rational combinatorial therapies for the effective treatment of bladder 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 1650.
Abstract 1652: Global proteomics analysis by SILAC (stable isotope labelling with amino acid in cell culture) in hepatocellular carcinoma treated by sorafenib: identification of phospho-AKT activation as a resistant mechanism
Authors: Feng Wen-Chi , Hsu Chih-Hung , Lin Shiou-Der , Gao Ming , Yeh Pei-Yen , Chow Lu-Ping , Cheng Ann-Lii
Published: February 2011

Sorafenib, a multikinase inhibitor against Raf kinase and vascular endothelial growth factor receptor (VEGFR), was recently approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the majority of HCC are inherently resistant to sorafenib. A global proteomic approach was applied to delineate the molecular mechanisms affecting the effectiveness of sorafenib in HCC cells. HCC cells (Huh7, SKHep1, and PLC5) were treated with sorafenib and analyzed by the SILAC (Stable Isotope Labeling with Amino acids in Cell culture) methodology. A total of 354 phosphoproteins were identified from untreated Huh7 cells (labeled with light amino acid, designated as L) and sorafenib-treated cells (labeled with heavy amino acid, designated as H). Forty-five proteins with high L/H ratio, indicating decreased expression after sorafenib treatment, were recognized. Among them, downstream proteins of Raf kinase, which are expected to be downregulated by sorafenib, were found. On the other hand, 30 proteins with low L/H ratio, indicating increased expression after sorafenib treatment, were recognized. Biological interaction network analysis of the phosphoproteome using IPA (ingenuity pathway analysis) method revealed that several signaling pathways including PI3K/AKT were significantly altered in sorafenib-treated Huh7 cells. To validate the relevance of these pathway alterations, the expression of p-AKT (Ser 473) was examined and was found significantly upregulated by sorafenib. Downregulation of AKT by siRNA attenuated the upregulation of p-AKT induced by sorafenib and enhanced the cytotoxicity of sorafenib in Huh7 cells. Combination of MK-2206, an allosteric inhibitor of AKT, abolished sorafenib-induced p-AKT upregulation, and resulted in a synergistic anti-proliferative effect in HCC cells. In conclusion, an unbiased and global phosphoproteomic approach by SILAC proves useful in identifying molecules altered by sorafenib in HCC cells; and by using this approach we showed that targeting AKT is a potentially useful strategy to improve the efficacy of sorafenib in advanced HCC. (The study was supported by the grant NSC 97-2628-B-002-004-MY3 from National Science Council of Taiwan and a research grant from Merck & Co., Inc.)

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 1652.
Abstract 1653: Highly effective treatment of HER2-amplified breast cancers through the complete inactivation of the HER2-HER3 tumorigenic driver
Authors: Amin Dhara N. , Sergina Natalia , Ahuja Deepika , McMahon Martin , Wang Donghui , Hann Byron , Shokat Kevan , Moasser Mark M.
Published: February 2011

Approximately 25% of breast cancers are driven by amplification of the HER2 oncogene and over-activity of the HER2-HER3 signaling complex. It is a promising hypothesis that the pharmacologic inactivation of HER2-HER3 signaling with tyrosine kinase inhibitors (TKIs) in patients will be a highly effective treatment for this disease. Treatment of HER2 amplified breast cancers with HER2 targeting agents shows that the HER2-HER3 complex is endowed with a two-log signal buffering capacity, making drug potency a critical and potentially limiting variable. The buffering capacity is mediated through Akt driven negative feedback signaling, which has the ability to amplify HER2-HER3 signaling and protect it against incomplete inhibition of HER2 kinase. The concomitant use of downstream inhibitors induces compound sensitizing and desensitizing mechanisms and incremental anti-tumor effects. The HER2-HER3 signal buffering mechanisms cannot ultimately compensate for the total loss of HER2 catalytic activity, and at fully inactivating doses of TKI, the failure to phosphorylate HER3 and the dual loss of MAPK and Akt pathway signaling leads to apoptotic tumor cell death. Such HER2-inactivating dosing is not tolerable in vivo using continuous schedules but is feasible and highly effective using intermittent dosing. We show that continuous dosing of the HER2 TKI, lapatinib, at its maximum tolerated dose in mice bearing HER2-amplified tumors produces modest growth inhibitory effects. However, a five day repeated intermittent schedule allows eight fold higher dosing and produces much more profound tumor regression and durable anti-tumor effects. Therefore high dose intermittent TKI therapy is an imminently testable clinical treatment hypothesis that can fully inactivate HER2-HER3 signaling by overpowering its signal buffering capacity, revealing a monotherapy potential in HER2-targeting TKIs that may surpass many of the combination therapy approaches currently being explored.

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 1653.
Abstract 1656: Id1, inhibitor of differentiation, is a key protein mediating anti-tumor responses of gamma-tocotrienol in breast cancer cells
Authors: Yap Wei Ney , Zaiden Norazean , Tan Yee Ling , Chang Piek Ngoh , Zhang Xue Wu , Wong Y.C , Ling M.T , Yap Yee Leng
Published: February 2011

Background: Gamma-tocotrienol has demonstrated anti-proliferative effect on breast cancer (BCa) cells, but mechanisms involved are largely unknown. This study aimed at deciphering the molecular pathways responsible for its activity. Methods: MTT cell proliferation, DNA fragmentation, TUNEL assay and flow cytometry were performed to investigate gamma-tocotrienol induced cell cytotoxicity and cell cycle arrest. Western blotting was used to characterize multiple signaling pathways associated with gamma-tocotrienol in BCa. Docetaxel, a chemotherapeutic drug that is able to provide significant survival advantage in metastatic BCa, was used in the chemosensitization studies. Results: Our results showed that treatment of BCa cells with gamma-tocotrienol resulted in induction of apoptosis as evidenced by activation of pro-caspases (cleaved caspase-3, −7, −8, −9 and cleaved PARP), accumulation of sub-G1 cells and DNA fragmentations. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of Id1 and NF-κB through modulation of their upstream regulators (Src, Smad1/5/8, Fak and LOX). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK signaling pathway and inhibition of JNK activity by specific inhibitor partially blocked the effect of gamma-tocotrienol. Furthermore, synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Interestingly, in cells that treated with gamma-tocotrienol, alpha-tocopherol or ß-aminoproprionitrile were found to partially restore Id1 expression. Meanwhile, this restoration of Id1 was found to protect the cells from gamma-tocotrienol induced apoptosis. Consistent outcome was observed in cells ectopically transfected with the Id-1 gene. Conclusion: Our results suggested that the anti-proliferative and chemosensitization effect of gamma-tocotrienol on BCa cells may be mediated through downregulation of Id1 protein.

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 1656.
Abstract 1657: HSP90 inhibition down regulates EGFR and its effector signaling proteins in pancreatic cancer cell lines
Authors: Chandana Sreenivasa R. , Leece Cheryl , Bhutani Manisha , Conley Barbara A.
Published: February 2011

Rationale: Currently approved therapies, including EGFR inhibitors, have minimal effect on survival in pancreatic cancer (PC). HSP90 is a molecular chaperone that specifically enables activity of numerous protein kinases, including EGFR and other signaling proteins. We have reported that simultaneous inhibition of EGFR and HSP90 may be additive in inhibiting cell viability in PC cell lines. Here we report possible molecular mechanisms underlying HSP90 and EGFR inhibition. Methods: MiaPaCa-2 and PANC-1 were treated with EGFR inhibitor, compound 56 (CMP56) (5 − 35 μmol), and HSP90 inhibitor geldanamycin (250 nmol − 1μmol). Growth inhibition was evaluated using the MTT assay, and apoptosis was evaluated by caspase-3 and PARP cleavage. The effect on relevant cell signaling proteins was assessed by immunoblotting with specific antibodies directed against EGFR and effectors such as AKT, STAT-3 and JNK. Results: Growth inhibition was significantly (p < 0.05) higher in both MiaPaCa-2 and PANC1 cells treated with CMP56 and geldanamycin than in cells treated with either agent at various concentrations. The molecular effects are shown in table 1. There was a decrease in phosphorylation of EGFR in bothe cell lines after treatment with CMP56. Concomitant inhibition of EGFR and HSP90 was associated with decreased phosphorylation of ERK, JNK and AKT and with an increase in caspase-3, PARP cleavage in MiaPaCa-2 cells, but not in PANC-1 cells. Conclusions: These results suggest that HSP90 inhibition in MiaPaCa-2 cells induces down regulation of EGFR, STAT-3 and AKT, but not in PANC-1 cells. Higher concentrations of geldanamycin may be required in PANC-1cells. Combined inhibition of EGFR and HSP90 induced apoptosis additively. We are studying further to understand the other potential molecular pathways involved in this strategy.Table.Molecular effects of EGFR and HSP90 inhibition in PC Cell lines ↘ - Decrease, - No change, GEL - Geldanamycin (500 nmol), CMP-56 - Compound 56 (10 μmol)Protein of interest MiaPaCa-2   PANC-1  GELCMP56GEL + CMP56 GELCMP56GEL + CMP56EGFR↘-↘↘ --↘AKT↘-↘↘ --↘STAT-3↘-↘↘ --↘

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 1657.
Abstract 1661: Effect of thalidomide and liposomal doxorubicin on human osteoclastogenesis
Authors: Chen Yu-Jen , Chen Yu-Yawn , Liao Hui-Fen , Chi Chih-Wen
Published: February 2011

Background: Thalidomide has been reported capable of inhibiting angiogenesis and growth of multiple myeloma. Liposomal doxorubicin is known to be mainly absorbed and degraded in reticuloendothelial system, especially the monocyte-macrophage lineage. Since osteoclasts (OCs) are derived from precursors of monocyte-macrophage lineage, we aimed to examine the effect of thalidomide, liposomal doxorubicin and their combination on human osteoclastogenesis.

Materials and Methods: We isolated CD14+ cells from peripheral blood mononuclear cells of healthy subjects and generated OCs under stimulation with macrophage-colony forming factor and receptor activator of NK-κB ligand. Cell viability, surface CD51/61 expression, and tartrate-resistant acid phosphatase (TRAP) activity were assessed by using MTT, flow cytometry and immunohistochemical staining, respectively.

Results: In comparison with control differentiated OCs, combination of thalidomide (purchased from TTY Biopharm, Taiwan) and liposomal doxorubicin (purchased from TTY Biopharm, Taiwan) profoundly inhibited the amount of harvested viable OCs. By treatment with each drug alone, thalidomide increased and liposomal doxorubicin decreased the amount of viable OCs. The expression of osteoclast-specific surface antigen CD51/61 was greatly inhibited by each drug and their combination. Furthermore, the amount of multinucleated TRAP-positive cells characteristics of OCs, was suppressed by each drug and, the mostly, their combination. Elucidation of cellular and molecular mechanisms of action has been undergoing.

Conclusion: Thalidomide, liposomal doxorubicin and their combination may effectively inhibit human osteoclastogenesis.

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 1661.
Abstract 1668: Pharmacodynamic biomarker development for INK128, a potent and selective inhibitor of TORC1/2 for the treatment of cancer
Authors: Jessen Katayoun , Wang Shunyou , Guo Xin , Kessler Linda , Kucharski Jeff , Staunton Jocelyn , Lan Lucy , Elia Marikka , Stewart Josh , Brown Jolene , Li Liansheng , Chan Katrina , Martin Michael , Ren Pingda , Rommel Christian , Liu Yi
Published: February 2011

Pharmacokinetic / pharmacodynamic (PK/PD) modeling is a valuable strategy to achieve target inhibition as well as predictable and meaningful therapeutic efficacy. The mammalian target of rapamycin (mTOR) comprises two protein complexes, TORC1 and TORC2, which together regulate cell growth, metabolism, angiogenesis, and cell survival. Because TORC1 and TORC2 play a crucial role in several pathways that are frequently dysregulated in human cancer, the mTOR kinase is a compelling target for oncology drug development. Through rational drug design we have identified INK128, a potent and selective small molecule, ATP-competitive, active-site TORC1/2 kinase inhibitor with excellent drug-like properties. Inhibition of phosphorylation of S6 and 4EBP1, downstream markers of TORC1 signaling, was selected for PD analysis in peripheral blood cells (PBCs), skin tissue, and tumor tissue biopsy in mice xenograft tumor models. Time- and dose-dependent inhibition of S6 and 4EBP1 was demonstrated in PBCs by phospho-flow (FACS) analysis. Immunohistochemistry and immunoblot analysis demonstrated a correlation between S6 and 4EBP1 inhibition in tumors or skin tissue and antitumor effect. Additionally, site-selective inhibition of AKT phosphorylation at Ser473, the downstream substrate of TORC2, was also demonstrated in tumors and skin biopsies in mouse xenograft models. Our results demonstrate that daily, oral administration of INK128 selectively inhibits PI3K/AKT/mTOR signaling at the level of TORC1/2, and show that INK128 inhibits growth, and in some cases induces regression, of various tumor xenograft models. Results from these studies display a clear pharmacokinetic and pharmacodynamic relationship. Moreover, the activity of several of these downstream markers can be reproducibly measured in human peripheral blood cells and may permit development of a PK/PD model that might assist to predict PBC and skin tissue PD marker inhibition time-profiles in patients. In summary, INK128 presents a compelling, biomarker-guided approach for the treatment of a variety of cancer by targeting TORC1/2 signaling.

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 1668.
Abstract 1676: Cytotoxicity of fenretinide and its metabolite 4-oxo-4-HPR in malignant lymphoid cells can be dependent and independent of reactive oxygen species (ROS)
Authors: Cooper Jason P. , Reynolds Charles P. , Curley Robert W. , Kang Min H.
Published: February 2011

Fenretinide (4-HPR) is a cytotoxic retinoid that increases ROS and ceramides and has shown clinical activity against neuroblastoma and lymphoid malignancies. We compared the cytotoxicity, ROS generation, and sphingolipid synthesis of 4-oxo-4-HPR (a recently identified 4-HPR metabolite) with 4-HPR in malignant lymphoid cells. Cytotoxicity of 4-oxo-4-HPR and 4-HPR was measured in 14 malignant lymphoid cell lines using a DIMSCAN system. Median concentrations cytotoxic for 90% of cells (IC90) were 2.1 µM (range: 1.1 − 15.9) for 4-oxo-4-HPR and 4.4 µM (range: 0.7 − 29.2) for 4-HPR. In 2 of 3 cell lines tested, ROS generation (DCFDA by flow cytometry) was higher with 4-HPR vs. 4-oxo-4-HPR (table). Ascorbic acid (AA) abrogated ROS generation and significantly attenuated cytotoxicity of 4-oxo-4-HPR and 4-HPR in CCRF-CEM and COG-LL-317 cells. However, in GA-10 cells AA completely abrogated ROS generation, but not cytotoxicity, for both 4-oxo-4-HPR and 4-HPR. Intracellular sphingolipid (SL) changes, another suggested mechanism for 4-HPR cytotoxicity, were measured by tandem mass spectrometry in GA-10 cells at 48-hours, and both 4-oxo-4-HPR and 4-HPR treatment increased SL, with significantly higher (p < 0.05) increases by 4-oxo-4-HPR. The fold increase/control for 4-oxo-4-HPR vs. 4-HPR: ceramides = 2.1 ± 0.13 vs. 1.3 ± 0.11; dihydroceramides = 87.6 ± 7.1 vs. 34.0 ± 5.1; sphingomyelins = 4.1 ± 0.14 vs. 2.4 ± 0.04). Thus, 4-oxo-4-HPR is similar in cytotoxicity to 4-HPR, less active in generating ROS, but more active in increasing SL. The cytotoxicity of both 4-HPR and 4-oxo-4-HPR in the GA-10 cell line is independent of ROS, providing a novel in vitro model for identifying the role of SL in the cytotoxicity of these agents.Table.Cytotoxicity and ROS generation for 4-HPR and 4-oxo-4-HPR, and cell survival after ROS abrogation. Cell Line (Histopathology)CCRF-CEM (T ALL)COG-LL-317 (T ALL)GA-10 (B Burkitt lymphoma)IC90 (µM)4-oxo-4-HPR1.32.41.54-HPR1.01.57.3     ROS Generation‡ (fold increase/control)4-oxo-4-HPR2.3 ± 0.03***1.6 ± 0.02***1.7 ± 0.04**4-HPR8.3 ± 0.36***5.3 ± 0.26***2.2 ± 0.29**     ROS abrogation‡ (% inhibition from drug alone)4-oxo-4-HPR + AA55%***31%*100%*4-HPR + AA83%***91%***100%***     Cell Survival at 20 hours‡AA102.5% ± 12.6107.3% ± 14.986.8% ± 14.94-oxo-4-HPR12.9% ± 2.926.3%± 2.010.9% ± 6.84-oxo-4-HPR + AA22.9% ± 3.5***63.1% ± 8.6***9.1% ± 3.74-HPR1.2% ± 0.69.1% ± 2.011.9% ± 6.24-HPR + AA27.9% ± 6.8***91.2% ± 11.4***12.6% ± 3.1‡ Treatments: 10 µM 4-oxo-4-HPR, 10 µM 4-HPR, 200 µM AA; * P < 0.05, ** P < 0.01, *** P < 0.001

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 1676.
Abstract 1680: Characterization of olanzapine glucuronidation: Potential role of the UGT2B10 codon 67 SNP in olanzapine metabolism
Authors: Erickson Kathryn K. , Lazarus Philip
Published: February 2011

Olanzapine (OLZ) is an atypical antipsychotic with broad applications for cancer patients including treatment-resistant depression, psychiatric problems such as schizophrenia or bipolar disorder, and chemotherapy-induced nausea and vomiting. The half-life of OLZ varies widely between patients. The purpose of the present study was to determine the relative contribution of various UDP-glucuronosyltransferases (UGTs) that are known to metabolize OLZ and examine the role of functional UGT genetic variants as a potential determinant of OLZ metabolism.

OLZ glucuronides were identified by ultra-pressure liquid chromatography (UPLC) and the UGTs exhibiting activity against OLZ were determined using homogenates of UGT-over-expressing HEK293 cell lines. Kinetic analysis was performed for active wild-type and variant UGTs. The clinical and physiological relevance of polymorphic active UGTs was examined by studying OLZ metabolism in 114 human liver microsomes and characterizing their glucuronidation activity via UPLC. One-Way ANOVA using Tukey's family error rate was used for pairwise comparisons between cell lines. Student's 2-sample t-test was used to compare human liver microsome glucuronidation data.

UPLC analysis of cell line homogenate samples revealed three distinct glucuronidation products. We found that two UGT enzymes, UGT1A4 and UGT2B10, exhibit glucuronidation activity against OLZ. The enzymatic activity of UGT1A4 codon 24 and codon 48 variants against OLZ was significantly different from wild-type UGT1A4 (both p < 0.05). In addition, the UGT2B1067Tyr variant exhibited no glucuronidation activity against OLZ. Human liver microsomes homozygous for the UGT2B1067Tyr variant exhibited an 82% reduction in glucuronidation activity against OLZ (p<0.05).

These data demonstrate the significance of a previously unrecognized UGT (UGT2B10) in OLZ excretion and the potential importance of a functional SNP at codon 67 of the UGT2B10 gene in the wide variability observed in OLZ metabolism between individuals. This could have an important impact on overall patient response/toxicity to OLZ.

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 1680.
Abstract 1683: ATF4 down-regulation potentiates L-asparaginase activity in ovarian cancer cell lines
Authors: Lorenzi Philip L. , Llamas Jenny , Caplen Natasha J. , Weinstein John N.
Published: February 2011

The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synthetase (ASNS) is strongly correlated with L-ASP activity in leukemia cell lines of the NCI-60 cell line panel. Unexpectedly, however, the correlation was also apparent in ovarian cancer lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. Using siRNA to down-regulate ASNS by 5-fold produced a proportional ∼5-fold potentiation of L-ASP activity in some ovarian cancer lines (OVCAR-3 and OVCAR-4). Surprisingly, ASNS siRNAs yielded a striking >500-fold potentiation in other ovarian lines (OVCAR-8 and OVCAR-8/ADR). Taken together, those findings suggest that the L-ASP/ASNS correlation is causal but also support the hypothesis that L-ASP activity is modulated by additional molecules or pathways. To address that hypothesis and to further elucidate the L-ASP mechanism of action, we have initiated genome-wide siRNA library screening. Preliminary screens in the OVCAR-8 cell line using a 418-gene apoptosis library, a custom 392-gene metabolic library, and the MTS assay as an endpoint yielded Z′ factors >0.90, indicating a robust screen. RNAi-mediated down-regulation of apoptosis genes had little or no effect on L-ASP's anticancer activity. That was surprising because L-ASP has been reported to cause apoptosis through glutamine depletion. Knock-down of ATF4, a transcription factor involved in cell metabolism, did markedly potentiate L-ASP activity (>500-fold). Furthermore, ATF4 knock-down markedly enhanced L-ASP anticancer activity toward the highly resistant cell line, OVCAR-4. These findings suggest that ATF4 is an important mediator of L-ASP resistance and provide rationale for investigation of ATF4 as a predictive biomarker of L-ASP effect.

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 1683.
Abstract 1688: Effect of Gefitinib on the reactions of mammalian type I and type II DNA topoisomerases
Authors: Topcu Zeki , Unlukurt Isa , Zencir Sevil
Published: February 2011

Developing small molecule inhibitors in anti-cancer drug research is an integral part of clinical oncology. Gefitinib (Iressa) is a promising small molecule inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. It was shown to have the potential to improve the clinical effectiveness of EGFR-targeted therapies and gained a significant impact in treatment of non-small-cell lung cancer (NSCLC). Over the past years, several naturally occurring and synthetic compounds with anti-cancer activities were shown to exert their actions via interfering with normal DNA topoisomerase reactions. DNA topoisomerases are ubiquitous enzymes that regulate DNA topology by making transient breakages at nucleic acid backbone and rejoining of DNA strands. Topoisomerases carry out their reactions by two different mechanisms; Type I topoisomerases make a single-stranded break in a DNA duplex while type II topoisomerases create transient breaks in both strands of a duplex. In this study we aimed to examine the effects of Gefitinib on these enzymes to extend the evaluation of its biological activities by plasmid supercoil relaxation and kinetoplast decatenation assays for type I and type II topoisomerases, respectively. Our results are discussed in relation to the outcomes of Gefitinib reported in clinical trials, which together would contribute in identifying the mechanisms of Gefitinib-induced tumor cell-killing.

Key Words; Gefitinib; DNA topoisomerases; anti-cancer drugs

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 1688.
Abstract 1689: Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis for 7-carboxymethylguanine and 7-carboxyethylguanine in human liver DNA
Authors: Wang Mingyao , Cheng Guang , Chen Li , Villalta Peter W. , Hecht Stephen S.
Published: February 2011

The carcinogenic nitrosamines N-nitrososarcosine (NSAR) and 3-(methylnitrosamino)propionic acid (MNPA) have been detected in the diet, tobacco products, and human urine. Metabolic activation of NSAR by methyl hydroxylation would produce a DNA carboxymethylating agent while similar metabolism of MNPA would produce a carboxyethylating agent, each of which would be expected to react at the 7 position of guanine to give 7-carboxymethylguanine (7-CMG) and 7-carboxyethylguanine (7-CEG), respectively. Furthermore, acrylic acid, a major industrial product, environmental contaminant and likely endogenous metabolite, and β-propiolactone, are also known to react with DNA to give 7-CEG. We developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS-SRM) method to quantify these adducts in human liver DNA. [15N5]7-CMG and [15N5]7-CEG were synthesized and used as internal standards. After addition of the internal standards, the DNA was hydrolyzed enzymatically and the hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-CMG and 7-CEG was treated with acetyl chloride in methanol to convert them to the corresponding methyl esters. After re-purification by solid-phase extraction, the appropriate fraction was analyzed by LC-ESI-MS/MS-SRM for the transitions m/z 224[M + H]+ → m/z 164[(M + H) - C2H4O2]+ for 7-CMG and m/z 238[M + H]+ → m/z 152 [BH]+ for 7-CEG. Human liver DNA samples had clear peaks co-eluting with [15N5]7-CEG, but no peaks were observed at the retention time of 7-CMG. The on column detection limits were approximately 10 and 5 fmol, respectively, for the methyl esters of 7-CMG and 7-CEG. The detection limit for the methyl ester of 7-CEG in DNA (starting with 1 mg) was approximately 8 fmol. Known addition studies using 7-CEG and calf thymus DNA demonstrated that the method was accurate and precise. Artifactual formation of 7-CEG during DNA isolation and enzyme hydrolysis was excluded. The method was applied for the analysis of 27 human liver samples. 7-CEG, but not 7-CMG, was detected in each sample, mean ± S.D, 408 ± 375 fmol/µmol guanine (82 adducts per 109 nucleotides), range 17 − 1491 fmol/µmol guanine. This is the first report demonstrating the presence of 7-CEG in DNA from humans. While MNPA, β-propiolactone and acrylic acid are possible sources of this DNA adduct, we consider acrylic acid to be the most likely because of its ubiquitous occurrence and probable endogenous formation from the lipid peroxidation product acrolein.

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 1689.
Abstract 1690: Analysis of benzo[a]pyrene and phenanthrene tetraol enantiomers in human urine: Relevance to the bay region diol epoxide hypothesis of benzo[a]pyrene carcinogenesis and to biomarker studies
Authors: Hecht Stephen S. , Carmella Steven G. , Hochalter J. Bradley
Published: February 2011

One widely accepted metabolic activation pathway of the prototypic carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) proceeds through the bay region diol epoxide BaP-(7R,8S)-diol-(9S,10R)-epoxide. However, few studies have addressed the analysis of human urinary metabolites of BaP which result from this pathway. Phenanthrene (Phe) is structurally related to BaP, but human exposure to Phe is far greater and its metabolites can be readily detected in human urine. Thus, we have proposed that Phe metabolites can be biomarkers of PAH exposure and metabolic activation. Phe-tetraols in particular could be biomarkers of the diol epoxide pathway. While BaP-tetraols and Phe-tetraols have been previously quantified in human urine, no published studies have determined their enantiomeric composition. This is important because different enantiomers would result from the bay region diol epoxide and “reverse” diol epoxide pathways, the latter being associated with weak mutagenicity and carcinogenicity. We addressed this problem using chiral HPLC to separate the enantiomers of Phe-1,2,3,4-tetraol and BaP-7,8,9,10-tetraol. Urine samples from smokers were subjected to hydrolysis with β-glucuronidase and arylsulfatase followed by solid-phase extraction on a Strata-X polymeric sorbent cartridge and a Bond Elute phenylboronic acid cartridge, chiral HPLC on a Cyclobond II or Pirkle column, and quantitation by gas-chromatography-negative ion chemical ionization-tandem mass spectrometry-selected reaction monitoring analysis of the Phe-tetraols, as their tetra-trimethylsilyl ethers. The results demonstrated that >96% of Phe-tetraol in smokers’ urine was Phe-(1S,2R,3S,4R)-tetraol, resulting from the “reverse” diol epoxide pathway, whereas less than 4% resulted from the bay region diol epoxide pathway of Phe metabolism. The corresponding (R,R)-stereochemistry of their precursor diols Phe-1,2-diol and Phe-3,4-diol was also confirmed. Urine from creosote workers with very high exposure to PAH was similarly analyzed for BaP-tetraol enantiomers. In contrast to the results of the Phe-tetraol analyses in smokers’ urine, >75% of BaP-tetraol in these human urine samples was BaP-(7R,8S,9R,10S)-tetraol resulting from the bay region diol epoxide pathway of BaP metabolism. These results provide the first direct support based on human urinary metabolite analysis for the bay region diol epoxide pathway of BaP metabolism and demonstrate differences in BaP and Phe metabolism which may be important when considering Phe-tetraols as biomarkers of PAH metabolic activation.

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 1690.
Abstract 1691: Apoptotic and antitumor effects of rotenone in MCF-7 human breast cancer cells are mediated ROS through activating JNK and p38 MAPK signaling
Authors: Lin Jen-Kun , Deng Yea-Tzy , Huang Hsiu-Chen
Published: February 2011

Rotenone is a natural hydrophobic pesticide derived from the roots of Derris and Lonchorcarpus species. The pesticidal activity of rotenone is attributed to irreversible binding and inactivation of complex of the mitochondrial transport chain. This can block electron transport from complex to ubiquinone, resulting in a blockage of the oxidative phosphorylation process, and an increase of the reactive oxygen species (ROS). Rotenone has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism is still not fully elucidated. In the present study, rotenone showed a strong growth inhibitory effect against human breast MCF-7 cells. DNA flow cytometric analysis, chromatin condensation and poly(ADP-ribose) polymerase (PARP) cleavage indicated rotenone actively induced apoptosis in MCF-7 cells. The anti-apoptotic protein BCl-2, was decreased, whereas the apoptotic protein, Bax, was increased in a time-dependent manner in rotenone-induced apoptosis. Moreover. The treatment of rotenone in MCF-7 cells caused the activation of c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPKs) and the inactivation of extracellular signal-regulated protein kinase1/2 (ERK1/2). The pharmacological inhibitor of JNK and p38 MAPK revealed significant protection against rotenone-induced apoptosis.

In summary, these results indicate rotenone may induce apoptosis through ROS and JNK/p38MAPK activation in MCF-7 cells. In conclusion, we examines the molecular mechanism involved in rotenone-induced apoptosis in human breast cancer MCF-7 cells. We have demonstrated a sequential events that rotenone-induced apoptosis in the cells through ROS generation, which serves as a pivotal upstream function in activating JNK and p38MAPKs, which leads to a decrease of BCl-2 and increase of Bax and finally to inducing apoptosis.

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 1691.