bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1325: PCI-5002 leads to enhanced radiosensitization via induction of necrotic cell death by promoting autophagy in non-small cell lung tumor xenograft model
Authors: Jung Dae Kwang , kim kwang woon , Lu Bo
Published: February 2011

Purpose: We previously showed that Bax/Bak inhibition induces autophagy and promotes radiosensitivity in vitro and in vivo. Therefore, we further investigated the specific mechanism by which radiation triggers necrotic cell death via autophagy in H460 cells treated with PCI-5002, water-soluble zinc ionophores, in vitro or in vivo.

Methods: Clonogenic assay was used to determine radiosensitivity. H460 lung cancer xenografts was generated and immunohistochemistry (IHC) was performed on tumor sections for tumor cell proliferation (Ki67 staining), angiogenesis (von Willebrand Factor staining), and apoptosis (cleaved caspase-3 antibody staining). Autophagy was determined by p62 protein, which is named sequestosome 1(SQSTM1) and links polyubiquitinated proteins to the autophagic machinery in vivo and punctuate localization of GFP-LC3 fusion protein, which observed under a confocal fluorescence microscope in vitro. Necrosis was determined with HMGB1 release and Propidium lodide (PI) staining.

Results: The PCI-5002 markedly enhanced sensitivity of MEF WT cells to radiation (DER=1.43, p=0.003) in clonogenic assay. This enhanced radiosensitization was associated with an increase in characteristic punctuate localization of GFP-LC3 when was blocked a cleaved caspase-3 in MEF WT cells treated with PCI-5002, suggesting that autophagy was induced simultaneously. We found that PCI-5002 with radiation showed a dramatically enhanced sensitivity of H460 cells to radiation (DER=1.92, p=0.001) in clonogenic assay, and tumor growth delay in a mouse hind limb xenograft model in vivo, and was well tolerated compared to control. In addition, the concurrent administration of PCI-5002 and radiation in vivo significantly decreased cell proliferation and vascular density compared to radiation alone. Consistent with the in vitro results, treatment both PCI-5002 and radiation showed a dramatically increase in HMGB1 as a marker for Necrosis and a significant decrease in p62 protein level for detecting autophagic flux in the lung tumor xenograft model.

Conclusions: These results suggest that induction of necrosis via promotion of autophagy may be utilized as a novel strategy to enhance radiation therapy in lung 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 1325.
Abstract 133: Effects of iASPP on paclitaxel-mediated mitotic catastrophe and chemoresistance in ovarian cancers
Authors: Jiang Li Li , Siu Michelle KY , Wong Oscar GW , Tam Kai Fai , Lu Xin , Lam Eric WF , Ngan Hextan YS , Le Xiao Feng , Wong Esther SY , Monteiro Lara J. , Chan Hoi Yan , Cheung Annie NY
Published: February 2011

Purpose: In this study, for the first time, we aimed to investigate the expression profile of iASPP in ovarian epithelial tumor in association with clinicopathological parameters. In vitro effect of iASPP on paclitaxel chemosensitivity will also be investigated.

Materials and Methods: Expression and amplification status of iASPP was examined in 140 clinical samples and 16 ovarian cell lines using immunohistochemistry, immunoblotting and quantitative real-time PCR, and correlated with clinicopathological parameters. Changes in proliferation, mitotic catastrophe and apoptosis in ovarian cancer cell lines of different p53 status following paclitaxel exposure were also analyzed using TUNEL assay, MTT assay and immunofluorescence.

Results: By immunohistochemistry and quantitative PCR, significantly higher protein and mRNA levels of iASPP was found in ovarian cancers when compared with borderline and benign ovarian tumour samples. Higher iASPP expression was also found in ovarian cancer cell lines than normal ovarian epithelial cell lines. Higher iASPP expression was significantly associated with carboplatin and paclitaxel chemoresistance (P=0.04), shorter overall (P=0.003) and disease-free survival (P=0.001). Multivariate analysis confirmed iASPP expression as an independent prognostic factor. Increased iASPP mRNA expression was significantly correlated with its gene amplification (P=0.018), suggesting that gene amplification contributes to overexpression of iASPP in ovarian caner. By in vitro assay, we demonstrated that iASPP overexpression in ovarian cancer cells conferred resistance to paclitaxel by reducing mitotic catastrophe in a p53-independent manner via activation of separase whilst knockdown of iASPP enhanced paclitaxel-mediated mitotic catastrophe through inactivating separase. Both securin and cyclin B1/CDK1complex were involved in regulating separase by iASPP. Conversely, overexpressed iASPP inhibited apoptosis in a p53-dependent mode.

Conclusion: Our findings suggested that iASPP may be explored as a potential prognostic marker and a novel molecular target for chemotherapy of ovarian cancers, especially in patients with chemoresistant or recurrent cancers.

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 133.
Abstract 1365: Novel anti-angiogenic peptides as potential therapeutic agents in combination with chemotherapy for breast cancer
Authors: Rosca Elena V. , Koskimaki Jacob E. , Popel Aleksander S.
Published: February 2011

Breast cancer is one of the most commonly diagnosed malignancies in women. Despite the intensive ongoing research it is estimated that approximately 200,000 new diagnoses will be made in 2009 and 40,000 deaths will occur due to this disease. Angiogenesis, the development of vessels from pre-existing microvasculature, is an essential component of tumor progression and has emerged as a therapeutic target for anti-angiogenic therapies in cancer. The process is controlled by a balance between endogenous pro- and anti-angiogenic factors, and involves numerous interactions among several cell types including proliferation, invasion, and migration. Bevacizumab (Avastin, Roche/Genentech), an anti-VEGF monoclonal antibody, has been approved by the FDA for several types of cancer; however, in the case of breast cancer its effect on the overall survival is only marginal.

Our laboratory has established a systematic bioinformatics approach to identify short novel anti-angiogenic peptides from natural proteins. These peptides belong to different classes (i.e. thrombospondin containing domain, collagen IV, chemokines etc) and in vitro experiments demonstrated their anti-angiogenic potential through inhibition of proliferation and migration of human endothelial cells. Moreover, several peptides have demonstrated efficacy in suppressing tumor growth when delivered as daily intraperitoneal injections in a mouse orthotopic breast cancer model. The repression was associated with a decrease in tumor microvasculature and an increase in necrotic core of the tumors (Koskimaki et al., 2009). The top performing candidate (a 24 amino acid peptide belonging to the collagen IV family) was further modified to find the optimal sequence for possible translation to clinical applications. Several truncations and substitutions of cystines by amino alpha-butyric acid were developed. The candidates were screened in in vitro proliferation assays and the top performer (full sequence length with substituted cystines) was tested in vivo using the orthotopic breast cancer model. This candidate demonstrated no significant changes in activity from the parent peptide, however making it is more conducive for clinical translation. We also investigate its application in combination with a typical chemotherapeutic agent (paclitaxel) and confirmed no decrease in activity in comparison to single agent therapy. We have performed further characterization of the mode of action for this peptide to identify its target receptors; this enhances the translation potential of this candidate as an anti-angiogenic, non-VEGF targeting agent for therapy in 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 1365.
Abstract 1435: Functional expression of chemokine receptor, CCR10, in patients with gastric cancer
Authors: Bae Jae-Moon , Lee Jun Ho , Kook Myoung Cheorl
Published: February 2011

Objectives: There is increasing evidence that chemokines and chemokine receptors are causally involved in the metastasis of cancer. The aim of this study was to investigate the expression of chemokine receptors, CCR10, and its prognostic role in patients with gastric cancer.

Methods: We screened the expression of CCR10 in 12 gastric cancer cell lines using the semi-quantitative RT-PCR. The expression of CCR10 was confirmed using Western blot and flow cytometry analysis of twelve gastric cancer cell lines. The function of CCR10 was examined using migration and proliferation assays. Then the migratory response of CCR10 was blocked using blocking antibodies. Finally, the clinical significance of the chemokine receptors was explored using tissue microarray methods and immunohistochemical staining of specimens from 753 gastric cancer patients.

Results: We found that twelve out of twelve (100.0%) gastric carcinoma cell lines expressed a functional CCR10 for its ligand, chemokine CCL27 and CCL28, as demonstrated by the migration assays, and the migration was inhibited by anti-CCR10 antibodies. The clinical samples evaluated by immunohistochemical assay of tissue microarrays showed that CCR10-positive carcinoma cells were detected in 22 of 753 (2.9%) cases. We failed to show that the patients with CCR10-positive tumors had significantly poorer prognosis than did those with CCR10-negative tumors.

Conclusion: These results suggest that CCR10 and its ligands were associated with increased tumor migration in gastric 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 1435.
Abstract 151: Utilizing 3-D nuclear DNA methylation patterns for cell-based assays in epigenetic drug screening
Authors: Gertych Arkadiusz , Farkas Daniel L. , Tajbakhsh Jian
Published: February 2011

The reversibility of epigenetic modifications, such as cytosine methylation, has gained much attraction in cancer therapy, with an emphasis on the development of anti-cancer drugs with demethylating potential to primarily reestablish tumor- suppressor gene functionality. The interest in the development of novel epigenetic drugs has increased the demand for cell-based assays to evaluate drug performance in the important pre-clinical phases of drug screening. Ideally a demethylating drug should reduce hypermethylation of tumor-suppressor genes for their reactivation, without creating unwanted effects: i.e. the hypomethylation of heterochromatin, of which a large percentage occurs in repetitive elements and can lead to genome instability. We have recently developed 3D quantitative DNA Methylation Imaging (3D-qDMI), an image-cytometrical approach, that can measure spatial nuclear distributions of methylated cytosine (MeC) and global DNA (gDNA) in addition to the nuclear MeC load in a high-throughput and cell-by-cell fashion to verify unwanted effects such as increased heterochromatin hypomethylation and decondensation that involve large-scale chromatin reorganization visualized by high-resolution confocal microscopy. Our novel approach succeeded in tracking differential changes of higher-order DNA organization due to drug-induced demethylation on a genomic scale, in human and mouse cancer cells. Our study showed that the differential distribution patterns of these two classes of DNA have the potential to be utilized as signatures for the characterization of different demethylating agents such as 5-azacytidine and zebularine.

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 151.
Abstract 1632: Co-targeting mTOR and IGF-1R/IR results in synergistic activity against a broad array of tumor cell lines, independent of KRAS mutation status
Authors: Barr Sharon , Russo Suzanne , Buck Elizabeth , Epstein David , Miglarese Mark
Published: February 2011

The IGF-1 receptor (IGF-1R) and insulin receptor (IR) are known to couple via IRS-1 to the PI3K signaling axis. Upon activation, these receptor tyrosine kinases stimulate PI3K, initiating a cascade of ser/thr kinases, including mTOR. mTOR is associated with two complexes: mTORC1, rapamycin sensitive, and mTORC2, which does not bind rapamycin. Since mTOR functions downstream of IGF-1R and IR, it is thought that tumor cells sensitive to IGF-1R inhibition would also respond to mTORC1/mTORC2 inhibition. To test this hypothesis we evaluated the effects of OSI-906, a selective small molecule dual kinase inhibitor of IGF-1R and IR and OSI-027, a selective catalytic-site inhibitor of mTORC1/mTORC2, alone and in combination in cell proliferation assays using a broad array of tumor cell lines. Across multiple tumor types, cells with activating KRAS mutations were typically less sensitive to OSI-027 than those expressing wild-type KRAS. In contrast, mutant KRAS did not reduce sensitivity to OSI-906. Cell lines with activating mutations in PIK3CA or loss of PTEN were sensitive to OSI-027 but the presence of a concurrent mutation in KRAS decreased their sensitivity to OSI-027. In KRAS mutant cells, OSI-906 was more effective than OSI-027 at reducing phospho-AKT levels. Conversely, in tumor cell lines with wild-type KRAS cells, OSI-027 more effectively reduced phospho-AKT levels compared to OSI-906. Similar effects were observed in a pair of isogenic cell lines engineered to express either wild-type or mutant KRAS. The observation that tumor cell lines exhibit differential sensitivity to inhibitors of IGF-1R/IR or mTORC1/mTORC2 supports the concept that IGF-1R and IR can activate multiple pathways and are not limited to signaling through the mTOR axis. Also, mTOR is a central signaling hub which integrates signaling from multiple inputs, not only the IGF-1R/IR cascade. The combination of OSI-906 and OSI-027 synergistically inhibited the proliferation of cancer cell lines, including those expressing mutant KRAS. In some cases, the combination also resulted in synergistic induction of apoptosis. These data provide support for further exploration of the potential utility of combining OSI-906 (dual IGF-1R/IR inhibitor) with OSI-027 (dual mTORC1/mTORC2 inhibitor) for the treatment of human cancers, including those with activating mutations in KRAS.

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 1632.
Abstract 1723: Thiazolinediones (TZDs) mediate proliferation and cell cycle kinetics in ovarian cancer cells in a PPARγ-dependent and -independent manner
Authors: Al-Alem Linah F. , Southard Chase R. , Kilgore Michael W. , Curry Thomas E.
Published: February 2011

Ovarian cancer is the leading cause of death in women with gynecological malignancies and is often called the ‘silent killer’. Thus, new treatment targets are constantly being investigated. PPARγ (Peroxisome Proliferator Activated Receptor gamma) is a nuclear transcription factor that has been implicated in several diseases. However, little is known about PPARγ in ovarian cancer. Recently, PPARγ agonists such as the thiazolinediones (TZDs) have been studied for their potential use as a therapeutic treatment for cancer. In the present study, we investigated the effect of the four TZDs: Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio), on cell proliferation and PPARγ expression in an ovarian cancer cell line: Ovcar3. Treatment with 10 μM CGZ or TGZ for 24 hours decreased proliferation, whereas Rosi and Pio had no effect. We observed that this decrease in Ovcar3 cell proliferation is due to a higher fraction of cells in the G0/G1 stage of the cell cycle. In order to assess whether this was mediated by PPARγ, we investigated the effect of TGZ and CGZ on PPARγ mRNA, protein expression and activity. As Rosi and Pio had no effect on proliferation, only CGZ and TGZ were further investigated. CGZ and TGZ increased PPARγ mRNA expression in Ovcar3 cells (6.9 and 18.1 fold respectively). However, levels of PPARγ protein expression did not correlate with mRNA levels. Moreover, luciferase reporter assays showed that neither of the TZDs increased PPARγ activity. Ovcar3 cells were transfected with a PPARγ overexpression vector (WT) or a mutant dominant negative form of PPARγ (DN). Both CGZ and TGZ increased PPARγ activity when the receptor was over-expressed. Interestingly, the over-expression of WT in unstimulated cells caused an increase in reporter activity, indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. Cells exhibited no changes in proliferation when transfected with the WT or DN forms of PPARγ. Also, the effect on proliferation was not impacted when CGZ or TGZ were added to the WT or DN transfected cells. These findings demonstrate that PPARγ agonists, CGZ and TGZ, stimulate PPARγ expression. However, the decrease in cell proliferation and increased cell cycle arrest seems to be due to both PPARγ dependent and independent mechanisms. These data suggest that the non-PPAR targets of TZDs need to be further investigated. (Supported by NIH RO1 CA 95609-01, and NCRR-P20-RR15592).

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 1723.
Abstract 1996: Molecular stratification of clear cell renal cell carcinoma by consensus clustering reveals distinct subtypes and survival patterns
Authors: Brannon A. Rose , Reddy Anupama , Seiler Michael , Arreola Alexandra , Chen Shufen , Moore Dominic T. , Pruthi Raj S. , Wallen Eric M. , Nielsen Matthew E. , Liu Huiqing , Ljungberg Börje , Zhao Hongjuan , Brooks James D. , Ganesan Shridar , Bhanot Gyan , Rathmell W. Kimryn
Published: February 2011

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer and presents in the clinical setting with a heterogeneous and difficult to predict natural history. Previous research has suggested the existence of molecular subtypes of ccRCC, but these studies have failed to elucidate the pathways driving these subtypes or provide gene sets translatable to clinic. Given the common use of molecularly targeted therapies in the clinic, a better understanding of the underlying tumor biology would clearly augment treatment decisions. In this study, gene expression microarray data from 48 patients was analyzed with ConsensusCluster, software that uses unsupervised consensus clustering algorithms to identify the most optimal innate molecular subclasses. Without clinical or other classifying information, this analysis identified two distinct subtypes of ccRCC within the training set, labeled clear cell type A (ccA) and B (ccB). Based on the most well-defined arrays in each subtype, Logical Analysis of Data (LAD) defined a highly accurate set of 120 probes that could then be subsampled to classify additional tumors individually. The subclasses were substantiated in a validation dataset of 177 tumors and analyzed for clinical outcome, demonstrating that tumors designated ccA have markedly improved disease-specific survival compared to ccB (median survival of 8.6 vs. 2.0 years, p=0.002). When analyzed by both univariate and multivariate analysis, the classification schema independently associated with survival. Furthermore, gene set analysis of both datasets suggests that ccB tumors overexpress more immature and aggressive pathways, while ccA tumors overexpress classic ccRCC pathways of metabolism and angiogenesis. Therefore, using only patterns of gene expression, a small gene set was defined that classified ccRCC into two robust subclasses with distinct biological pathway expression and marked differences in clinical outcome. This classification schema, which is applicable to individual tumors, has implicit repercussions for influencing treatment decisions, defining biological mechanisms involved in ccRCC tumor progression, and directing future drug discovery.

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 1996.
Abstract 2079: Estrogen receptor alpha expression with miR-206 trans-acting regulation roles in breast cancer cell lines
Authors: Choi Young Eun , Song Junkyu , Jo Yong Hwa , Park Hyun Mi , Yoon Kyung-Sik ,   Department of Biochemistry and Molecular Biology(BK21 project), MRC for Bioreaction to ROS and Bio
Published: February 2011

Estrogen receptor-alpha (ER-α) is present in 65% of human breast tumors. Tamoxifen(Tam) was only effective in ER-α expressed breast cancer cell lines. Also, it was reported that miR-206 decreases endogenous ER-α mRNA and protein levels in human MCF-7 breast cancer cells. We expect that effect of invasion and apoptosis in breast cancer cells by regulating expression of miR-206. Hence, we demonstrated that trans-acting regulation roles by ER-α and miR-206 in breast cancer cell line. MCF-7(ER hyper-expressed), MDA-MB231(ER hypo-expressed), MCF-10A(ER hypo-expressed normal breast cell line) cells were targeted experiments. Cell viability was analyzed by dose-dependent Tamoxifen with MTT assay. Whole-cell extracts were observed for protein levels of ER-α, ERK, p38, p-ATM, ATM, p-chk2, chk2, p-p53, p53, cyclinD1 with western blotting. By using, ER-α, p38, miR206 activator/inhibitor and real-time PCR, we studied change of molecules in cell lines. Cell viability of MCF-7 cell was reduced about 85% of the cells were dead by 5, 7μM Tam. ERK phosphorylation was increased by dose-dependent Tam in both MCF-7 cell and by 7μM Tam treatment in MDA-MB231 cell. Also, p38 phosphorylation was raised by dose-dependent Tam in MCF-7 cells and MDA-MB231 cells. ATM, chk2, p53 that involved DNA damage molecules were not affect by dose-dependent Tam. CyclinD1 known as cell cycle G1 regulation molecule was decrease by effect of Tam. Also, miR-206 levels by real-time PCR were lessening in MCF-7 and increase in MDA-MB231 and MCF-10A. Specific molecules of MCF-7, MDA-MB231 and MCF-10A were influenced by ER-α, p38, miR-206 activator/inhibitor. ER-α regulation and miR-206 expression were shown to trans-activated roles in breast cancer cell lines. Therefore, inhibition of miR-206 was accompanying the hyper-sensitivity of anticancer drug Tamoxifen that mediated ER-α. Whereas, miR-206 over expressed put down the invasion properties of breast cancer. We suggest that these roles of miR-206 will take the strategy for valuable breast cancer therapy.

Keywords: ER-α, miR-206, Tamoxifen, breast cancer

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 2079.
Abstract 2104: MicroRNA-101 promotes estrogen independent growth and confers tamoxifen resistance in ER positive breast cancer cells
Authors: Sachdeva Mohit , Wu Hailong , Ru Peng , Hwang Larn , Trieu Vuong , Mo Yin Yaun
Published: February 2011

MicroRNAs function as key gene regulators capable of silencing gene expression at the posttranscriptional level. It is estimated that over 1/3 protein coding genes are under control of microRNAs. For example, estrogen receptor (ER) is negatively regulated by microRNAs such as miR-22 and miR-206, and a consequence of this suppression causes resistance to tamoxifen, a commonly used clinical anticancer drug for treatment of breast cancer. However, it is not clear whether microRNAs play any role in the estrogen independent growth, leading to intrinsic tamoxifen resistance. In this study, we use an in vivo selection system against a microRNA library. We first infect the ER positive MCF-7 cells with the pooled library and then orthotopically inject them into female nude mice without supplement of estrogen. As expected, MCF-7 cells infected with the library form tumors whereas those infected with vector control form no tumors. Further experiments show that a number of microRNAs including miR-101 are enriched. Importantly, miR-101 alone is able to promote estrogen independent growth and causes tamoxifen resistance. Unlike those microRNAs that confer tamoxifen resistance by targeting ER, miR-101 has no effect on ER expression or its activity. Of considerable interest, miR-101 suppresses cell growth in normal medium, but it promotes cell survival in estrogen free medium. Finally, we show that miR-101 is able to activate Akt in part due to suppression of Magi-2, a scaffold protein involved in regulation of pTEN activity. Hence, direct suppression of Magi-2 by miR-101 would provide a molecular link for miR-101-mediated activation of Akt through suppression of the pTEN activity. Taken together, these results demonstrate a novel function of miR-101. As a result, miR-101 may serve as a biomarker for the subpopulation of ER positive breast cancer patients who are intrinsically resistant to tamoxifen, and furthermore, it may serve as a novel therapeutic target for overcoming the resistance.

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 2104.
Abstract 2196: Genome-wide analysis of DNA methylation patterns in formaldehyde-fixed paraffin-embedded (FFPE) human tumor specimens
Authors: Brenet Fabienne , Socci Nicholas D. , You Daoqi , Cabal Rafael , Ladanyi Marc , Viale Agnes J. , Scandura Joseph M.
Published: February 2011

Formalin-fixed paraffin-embedded samples (FFPE) are in many case, the only available source of biological materiel available for translational studies. However the quantity and quality of nucleic acid extracted from FFPE samples is very often challenging (small amount and high level of degradation), which makes genomic analysis in general, and epigenomics analysis in particular, difficult to impossible. Here we are showing that we developed a robust, unbiased and cost effective method for genome-wide analysis of DNA methylation of FFPE DNA. Our method, named STAMP for Sequence Tag Analysis of Methylation Patterns, uses a recombinant protein containing a methyl-CpG binding domain (MBD) to precipitate methylated DNA fragments that are then identified by massively parallel sequencing using the SOLiD sequencer (ABI). We generate ∼15-million sequence tags per specimen and utilize an analytical platform comprised of custom-written R-language algorithms to study genomic DNA methylation patterns. We used this technology to compare the methylation patterns of matched fresh frozen (FF) and FFPE tumor specimens. Tissues had been stored for as long as 6 years. Because the STAMP method utilizes fragmented DNA as starting material we hypothesized that this method would be well-suited to the study of FFPE DNA methylation. Using this method, we found the methylation patterns of the FFPE and matched FF specimens were highly correlated. We successfully detected previously known methylated patterns and found hundreds of novel methylated regions. DNA methylation patterns were observed at all scales across the genome: from whole chromosomes to individual genes. We found that densely methylated elements are often highly conserved or closely associated with gene coding regions and promoters. We identified distinct patterns of DNA methylation surrounding the transcription start and termination sites of all genes. Genome-wide analyses of DNA methylation patterns of FFPE samples using the STAMP assay opens up the possibility to analyze large repositories of tumor specimens as well as samples that have been collected during routine clinical work and clinical trials. Large scales studies of DNA methylation using existing FFPE specimens linked to treatment outcomes may be rapidly translated to new epigenetic prognostic measures for cancer patients.

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 2196.
Abstract 2218: Whole exome DNA sequencing in human cancers of the digestive system
Authors: Wheeler David A. , Gingras Marie-Claude , Cotton Ronald , Guiteau Jacques , Hodges Sally , Wang Min , Wu Yuan-Qing , Newsham Irene , Nazareth Lynne V. , Fisher William E. , Goss John A. , Brunicardi Francis C. , Muzny Donna M. , Gibbs Richard A.
Published: February 2011

The onset and progression of cancer is driven by extensive rearrangement and mutation of the genome. The ever-expanding sequencing capacity ushered in by the second generation of DNA sequencers has enabled complete base-level description of the cancer genome and is therefore deepening our understanding of the etiology of the disease. We have combined a variety of approaches centered on next generation sequencing technologies aimed at comprehensive characterization of the cancer genome to define the key molecular lesions that drive tumor initiation and progression. These include targeted exonic sequencing to define coding sequence mutation, whole genome shotgun sequencing to define structural rearrangements, and cDNA sequencing to define alteration in expression levels, aberrant splicing and fusion transcripts. We have applied these approaches to pancreatic adenocarcinoma, hepatocellualr carcinoma and colorectal carcinoma. Results of genome sequencing have yielded an unprecedented picture of the rearrangements and mutations from analysis of read sequence and read coverage across the chromosomes. Evident in both the cancers is a dramatic reduction in SNP variation in the tumor genome compared to the germline from the same individual owing to extensive loss of heterozygosity. Superimposed on this are a few thousand mutations, some of which drive the tumor phenotype. The corresponding set of analog data generated by SNP-array microarray platforms provide validation of the central results from sequencing which leads to a new appreciation of the relationship between genetic change and the malignant phenotype.

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 2218.
Abstract 2225: Differential RNA splicing in high-risk neuroblastoma detected by affymetrix exon array and next-generation sequencing
Authors: Guo Xiang , Song Young , Badgett James , Chen Qing-Rong , Wei Jun , Khan Javed
Published: February 2011

RNA splicing is an important regulatory mechanism of gene expression providing various functions for specific cells including cancers. In this study, we investigated global alternative splicing pattern in neuroblastoma using Affymetrix exon array (HuEx) and deep sequencing approach (RNA-Seq). Exon array profiling was performed on 47 neuroblastoma samples in stage 1 and stage 4 with normal or amplified MYCN copy number (stage 1-, 4- and 4+). Principal component analysis demonstrated distinct splicing pattern in three groups of samples. In stage 4- compared with stage 1- tumors, differentially spliced candidate genes (n=355) had little overlap with genes showing whole gene expression changes (n=158), and most of them were involved in different biological processes. In contrast, a larger number of genes exhibited either exon-level splicing, gene-level expression, or both changes in stage 4+ versus stage 1- tumors. To validate exon array results, we performed transcriptome sequencing of selected samples with stage 1-, 4-, or 4+ neuroblastomas. The concept of splicing index was adapted to measure RNA splicing by short sequence reads. Both exon array and deep sequencing data demonstrated differential splicing of microtubule-associated genes, which may play an important role in enhanced genomic instabilities and tumor development. In conclusion, our results demonstrated a significant contribution of alternative or aberrant splicing to neuroblastoma development. The identification of differentially spliced genes and pathways in high risk neuroblastomas may be essential to the understanding of cancer biology and the discovery of diagnostic biomarkers or therapeutic targets in neuroblastoma.

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 2225.
Abstract 224: Antitumorigenic effect of synthetic C-substituted diindolyl methanes originating from cruciferous vegetables in KB human oral cancer cells through endoplasmic reticulum stress
Authors: Choi Eun-Sun , Shim Jung-Hyun , Cho Sung-Dae , Cho Nam-Pyo
Published: February 2011

Diindolylmethane (DIM), an isothiocyanates found in cruciferous vegetables, has been shown to have cancer chemopreventive effects. A series of synthetic C-substituted DIMs (C-DIMs) analogs was developed, including DIM-C-pPhtBu and DIM-C-pPhC6H5, which exhibited better inhibitory activity in cancer cells than DIM. This study examined the effects of C-DIMs on the growth of KB human oral cancer cells. DIM-C-pPhtBu and DIM-C-pPhC6H5 decreased the number of viable cells and induced caspase-dependent apoptosis. The apoptotic cell death was accompanied by a change in Bax/Bcl-2 ratio and damage to mitochondrial membrane potential through the induction of Death Receptor 5 and the cleavage of bid and caspase 8. Mechanistic studies demonstrated that the apoptotic cell death induced by DIM-C-pPhtBu and DIM-C-pPhC6H5 was mediated by endoplasmic reticulum (ER) stress. This provides the first evidence that synthetic C-DIMs originating from cruciferous vegetables induce apoptosis in KB cells via the ER stress pathway.

This study was supported by Bio R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (M10870050003-08N7005-00311) and National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008-331-E00260).

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 224.
Abstract 2282: Inhibition of vasculogenesis, but not angiogenesis, reduces tumor recurrence following irradiation
Authors: Brown Martin , Kioi Mitomu , Ahn G-one
Published: February 2011

Tumor blood vessels can derive from two sources: by angiogenesis, or the sprouting of endothelial cells from nearby blood vessels, and by vasculogenesis, which is produced by circulating cells. We have proposed that the radiation doses given in radiotherapy, will severely restrict local angiogenesis and thereby force tumor regrowth to rely on vasculogenesis. This involves the recruitment of proangiogenic circulating cells many of which are derived from the bone marrow. To investigate this hypothesis, we used the subcutaneously implanted FaDu human head and neck tumor and an orthotopic brain tumor using human U251 GBM cells.

We found that local tumor irradiation induced the influx of bone marrow derived cells (BMDCs) in a dose-dependent manner into both tumor models with most of this increase reflecting influx of CD11b+ myelomonocytes. We postulated that this influx of BMDCs was stimulated by increased tumor hypoxia and HIF-1 levels caused by radiation damage to the tumor vasculature. To determine HIF-1 activity in real-time in our brain implanted U251 GBM, we stably expressed the HIF-1 reporter construct 5HRE-luc in U251 cells and showed that HIF-1 activity increased rapidly starting at about two weeks following 15 Gy, in parallel to the increase in tumor hypoxia that we observed at this time.

To test our hypothesis that the increased HIF-1 levels were responsible for the increased influx of CD11b+ cells into the tumors, we used the HIF-1 inhibitor NSC-134754. When this was given daily for 2 weeks, starting immediately following irradiation, the increased tumor levels of CD11b+ monocytes observed after 15 Gy was abrogated. This treatment also prevented the regrowth of the irradiated tumors following irradiation.

As a further test of our hypothesis we determined the effect of inhibiting the interaction of SDF-1 with its receptor CXCR4 using the clinically approved drug AMD3100, which we infused starting immediately following irradiation. This had no effect on the growth of unirradiated tumors in the brain but completely inhibited the recurrence of the irradiated tumors following either a single dose of 15 Gy or the more clinically relevant scheme of 5 daily doses of 2 Gy. Inhibition of angiogenesis with DC101 increased tumor growth delay but did not prevent tumor recurrences.

We also showed that the recurrence of FaDu tumors locally irradiated with 20 Gy was prevented using a monoclonal neutralizing antibody against CD11b+ monocytes. This treatment did not increase the radiosensitivity of normal skin thereby providing a therapeutic gain.

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 2282.
Abstract 2289: MRJ(L) reverts mesenchymal phenotype and restricts malignancy by inhibiting Wnt/β-catenin signaling
Authors: Samant Rajeev S. , Mitra Aparna , Menezes Mitchell E. , Shevde Lalita A.
Published: February 2011

Expression of MRJ (Hsp40/DNAJB6) is lost in advanced infiltrating ductal carcinoma. We found that ectopic expression of the large isoform of MRJ, MRJ(L), is functionally capable of retarding tumor growth. Our continuing efforts towards understanding the effect of MRJ(L) expression on cancer cells revealed that MRJ(L) caused morphological changes indicative of epithelial-like appearance. It decreased the expression of several mesenchymal markers such as N-cadherin, Twist, Slug, vimentin and up regulated epithelial marker, Keratin 18. However, most noticeable was the loss of β-catenin protein upon MRJ(L) expression. Differential-proteomic analysis of the secretome showed that MRJ(L) expression elevated DKK1 (Dickkopf1), a secreted inhibitor of Wnt/β-catenin signaling. Increased DKK1 levels enhanced proteosome mediated degradation of β-catenin and reduced Wnt/β-catenin signaling as measured by the TCF/LEF reporter activity in MRJ(L) expressors. Furthermore we found that silencing MRJ(L) from immortalized mammary epithelial cell line, MCF10A, led to decreased DKK1 and E-cadherin levels, with concomitant increase of β-catenin protein. We also found that silencing DKK1 from the MRJ(L) overespressing cancer lines led to increased malignant behavior as measured by foci formation, growth in soft agar and wound healing capacity. Overall, our findings lead us to conclude that MRJ(L) maintains the epithelial phenotype and restricts malignant behavior of tumor cells by inhibiting Wnt/β-catenin 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 2289.
Abstract 2291: Downregulation of uPA/uPAR inhibits epithelial to mesenchymal transition (EMT) in intermittent hypoxic DAOY and D283 medulloblastoma cells
Authors: Gupta Reshu , Chetty Chandramu , Bhoopathi Praveen , Lakka Sajani S. , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S.
Published: February 2011

Medulloblastoma is the most common, rapidly growing tumor of the cerebellum, which arises in childhood and has a tendency to metastasize via cerebrospinal fluid (CSF) pathways. In addition to surgical resection, post-operative radiotherapy and chemotherapy are the mainstays in treating patients with medulloblastoma. However, these tumors remain among the most challenging pediatric brain tumors. Factors related to the tumor microenvironment are being recognized as fundamental to tumor progression, increased resistance and metastasis. Hypoxia, one of these factors, leads to epigenetic and genetic adaptation of clones and increased EMT, invasiveness and metastasis. Thus, hypoxia is closely associated with tumorigenesis and provides tumors the ability to outgrow the existing vasculature. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and is responsible for EMT, invasion, migration, and survival of hypoxic-conditioned medulloblastoma cells. We also show that Akt and SNAIL are activated under hypoxia, and this response is blocked by plasmid vector-mediated delivery of uPA/uPAR siRNA. The linkage of uPAR to Akt activation under hypoxia provides one mechanism by which uPAR may regulate SNAIL, and thus, promote EMT. The results presented here suggest that, in cells that express high levels of uPAR, induction and maintenance of EMT depends on simultaneous activation of numerous cell-signaling factors downstream of uPAR. This study provides valuable evidence of the anti-tumor efficacy of plasmid vector-mediated delivery of uPA/uPAR siRNA in hypoxic-conditioned medulloblastoma cells to target intermittent hypoxia-induced cell invasion, migration, angiogenesis and EMT and to achieve better therapeutic outcomes in the treatment of malignant medulloblastomas

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 2291.
Abstract 2315: Influence of epithelial to mesenchymal transition on viral oncolysis in non small cell lung cancer
Authors: Schmitt Anthony D. , Fuchs Bryan C. , McGinn Christopher , Kuroda Toshihiko , Goodwin Jonathn M. , Tanabe Kenneth K. , Lanuti Michael
Published: February 2011

Background: Epithelial to mesenchymal transition (EMT) is distinguished by the loss of epithelial cell junction proteins such as E-cadherin and the relative gain of mesenchymal markers such as vimentin. We hypothesized that EMT may play a role in resistance to viral oncolysis in non-small cell lung cancer (NSCLC). Methods: Multiple human NSCLC cell lines obtained from the American Tissue Culture Collection (H292, H358, H441, H1437, A549, H1703 and SW1573) were evaluated for cell surface expression of E-cadherin via Western blot. Vimentin and Zinc finger E-box binding-1 (Zeb-1) mRNA expression were measured in all of the NSCLC cell lines using RT-PCR to further analyze a molecular profile. Herpes viral infectivity was tested in vitro at varying multiplicity of infection (MOI) using both wild type Herpes (KOS) and an attenuated (ICP-6 defective) HSV-1 mutant (hrR3). Cell surface levels of nectin-1 and herpes virus entry mediator (HVEM) were measured by fluorescence-activating cell sorting (FACS). Results: Western Blots on cell lysates revealed the presence of E-cadherin in H292, H358, H441, and H1437. The cell lines SW1573 and H1703 lacked E-cadherin and expressed higher transcript levels of Vimentin and Zeb-1, suggesting a more mesenchymal phenotype. Cell lines harboring epithelial markers appeared more susceptible to viral oncolysis at low MOI compared to those with mesenchymal markers. The mesenchymal-phenotype cells were 10-1000 fold more resistant to both wild type HSV-1 and replication conditional hrR3, thus suggesting a cell-growth independent mechanism. Viral cytotoxicity did not appear to be Nectin-1 mediated since there was no differential cell surface expression in mesenchymal or epithelial phenotypes. Conclusion: Infectivity and viral oncolysis of NSCLC cell lines may be related to epithelial or mesenchymal phenotype. Nectin-1 does not appear to be the major receptor for viral infectivity in NSCLC. Current and future studies focus on ways to reverse EMT in NSCLC, thus rendering tumor cells more susceptible to viral oncolysis and perhaps chemotherapy.

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 2315.
Abstract 2342: Prognosis for patients with adenocarcinomas of lower esophagus, gastro-esophageal junction and cardia can be directly evaluated by immunohistochemistry of uPAR
Authors: Illemann Martin , Ovrebo Kjell , Skarstein Arne , Christensen Ib J. , Alpízar-Alpízar Warner , Helgeland Lars , Danø Keld , Nielsen Boye S. , Laerum Ole D.
Published: February 2011

Adenocarcinomas of lower esophagus, gastro-esophageal junction and cardia in humans are highly invasive tumors with poor prognosis. In order to elucidate proteolytic mechanisms for the invasive behavior, we have studied the localization of urokinase-type plasminogen activator receptor (uPAR) in 65 cases. uPAR was expressed in nearly all cases of invasive adenocarcinomas by populations of cancer cells, macrophages and myofibroblasts. In areas with local carcinoma in situ lesions (15 cases), or with Barrett's metaplasia (21 cases) adjacent to the tumor tissue, and in six control cases with Barrett's metaplasia only, no uPAR-immunoreactivity was found. High local expression of uPAR therefore appears to be a characteristic marker for invasive behavior in this tumor.

Using a simple scoring system for semi-quantitative estimation of uPAR-positivity on immmunohistochemically stained specimens, a significant association between high uPAR-score in cancer cells in the tumor core and short survival was found independent of tumor TNM-stage (HR=1.74, 95% CI: 1.05-2.88). High uPAR-score for each individual cell type (cancer cells, macrophages and myofibroblasts) was also associated with short survival. A diagnostic procedure for scoring of uPAR-positivity might be useful for this type of tumors. High local proteolytic activity mediated through the plasminogen activation system, with combined action of malignant and non-malignant accessory cells may be an important mechanism for the rapid progression and high mortality in this tumor type.

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 2342.