bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4693: High expression levels of NOXA are important for p53-mediated hypersensitivity in testicular germ cell tumor (TGCT) cells
Authors: Gutekunst Matthias , Oren Moshe , Weilbacher Andrea , Dengler Michael A. , Aulitzky Walter E. , van der Kuip Heiko
Published: June 2012

Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer such as NTERA-2D1 show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis is responsible for this hypersensitive phenotype. However, the role of the p53 pathway remains controversial.

Using RNA interference-mediated gene silencing, we systematically investigated the impact of DNA damage kinases upstream of p53, p53 itself, as well as apoptosis-related p53 targets on this hypersensitivity. Unexpectedly, neither knockdown of ATM, ATR, or DNA-PK alone nor ATM/ATR double knockdown or ATM/ATR/DNA-PK triple knockdown had any significant protective effect on survival of NTERA-2D1 cells upon Cisplatin. However, siRNA-mediated silencing of p53 was sufficient to completely abrogate hypersensitivity demonstrating that in these cells p53 acts preferentially as a pro-apoptotic factor. This is also supported by the finding that NTERA-2D1 cells were not only hypersensitive to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. Screening of different p53 targets revealed that the pro-apoptotic function of p53 was dependent on NOXA, since silencing of this p53 target almost completely mimicked the effect of the p53 knockdown. Importantly, both constitutive and Cisplatin-induced levels of NOXA were significantly reduced in resistant NTERA-2D1 cells obtained by either short-term differentiation or cultivation with increasing Cisplatin concentrations. Furthermore, comparison of a panel of p53 wildtype tumor cell lines revealed exceptionally high NOXA levels in the most sensitive cell line NTERA-2D1.

In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to pro-apoptotic functions of p53 mediated by exceptionally high levels of NOXA.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2011-4693
Abstract 470: Role of Fli1 in hematopoietic stem cell-derived fibroblast promotion of tumor cell migration and invasion
Authors: McDonald Lindsay T. , Neitzke Daniel J. , Findlay Victoria J. , Turner David P. , Watson Patricia M. , Watson Dennis K. , LaRue Amanda C.
Published: June 2012

Cells and paracrine factors of the tumor microenvironment play a central role in tumor angiogenesis, invasion, migration and proliferation, making the tumor microenvironment an exciting therapeutic target. Among the most prominent cell types in the tumor stroma are fibroblasts, termed carcinoma-associated fibroblasts (CAFs). We have identified a novel population of CAFs and CAF precursors (circulating fibroblast precursors, CFPs) that are of hematopoietic stem cell (HSC) origin. These cells preferentially migrate and differentiate in response to tumor and their inhibition results in decreased tumor size. While these studies have identified a novel HSC-derived CAF population, the mechanisms by which these cells promote tumorigenesis are unknown. Previous studies have indicated a critical role for Fli1, an Ets family transcription factor, in regulation of HSCs and differentiation/maturation of hematopoietic lineages. Additionally, loss of Fli1 has been associated with cancer progression. Based on these findings, we hypothesize that HSC-derived CFPs/CAFs directly affect tumor cell migration and invasion and that loss of Fli1 in CFPs/CAFs enhances these effects. To address this hypothesis, we examined the ability of HSC-derived fibroblast populations to affect tumor cell migration and invasion in vitro. Fibroblasts were established from peripheral blood and bone marrow of normal mice and mutant Fli1 mice expressing a truncated Fli1 protein (Fli1ΔCTA) lacking the carboxy-terminal regulatory (CTA) domain. The effects of conditioned media from these fibroblast populations on tumor cell migration and invasion were examined in chemotactic transwell migration assays and matrigel-based invasion assays, respectively. Data show that conditioned media from all HSC-derived fibroblast populations promoted migration and invasion of tumor cells versus control, and that this effect was significantly enhanced using conditioned media from fibroblasts derived from Fli1ΔCTA/Fli1ΔCTA mice. Ongoing studies are directed at identifying the mechanisms by which Fli1 target genes affect HSC-derived fibroblast modulation of tumor cell migration and invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2011-470
Abstract 4708: Preclinical development of an anti-miR-21 compound for hepatocellular carcinoma
Authors: Marcusson Eric G. , Balakrishnan Asha , Chang Aaron N. , Bhat Balkrishen , Goga Andrei
Published: June 2012

micro-RNA-21 is frequently over-expressed and has been shown to correlate with poor outcome in multiple cancer types. Its genomic locus is amplified in some cancer types and miR-21 is also transactivated by several known cancer drivers (e.g. STAT3 and Ras). We have used a publically available data set from 86 patients to show that miR-21 is over-expressed in hepatocellular carcinoma (HCC). The treatment options for HCC are limited, thus making it the third leading cause of cancer related deaths worldwide. Additional treatments for HCC are greatly needed.

We investigated the therapeutic potential of inhibiting miR-21 with chemically modified oligonucleotides known as anti-miRs using models of HCC, including a genetically engineered mouse model of HCC driven by an inducible, activated form of Ras. Short term treatment with the anti-miR led to a reduction in tumor formation. Furthermore, inhibition of miR-21 was clearly demonstrated by analysis of genome wide mRNA expression data from treated versus untreated tumors. This data showed that genes containing the mir-21 octamer sequence were the most significantly over-enriched among up-regulated genes compared to all of the other 65,535 possible octamers. We also determined the effect of long-term anti-miR-21 treatment on the survival in this mouse model of HCC. anti-miR-21 treated mice demonstrated a highly significant survival advantage (p<0.0001) over saline and control anti-miR treated mice. Our findings suggest that miR-21 is a promising candidate for the therapeutic intervention of liver cancer and further highlights the potential of anti-miR-mediated inhibition of microRNAs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2011-4708
Abstract 4731: Population specific microRNA expression and biological implications
Authors: Huang R. Stephanie , Gamazon Eric R. , Ziliak Dana , Zhang Wei , Im Hae Kyung , Wing Claudia , Duan Shiwei , Bleibel Wasim K. , Cox Nancy J. , Dolan M. Eileen
Published: June 2012

Population differences are commonly observed for many complex traits including response to chemotherapy. These differences may be attributed to the combined effect of socioeconomic, environmental, genetic and epigenetic factors. Indeed, genome-wide genetic and gene expression differences have been reported among different ethnic populations. Furthermore, we have previously reported population differences in cellular sensitivity to carboplatin, daunorubicin and ara-C and have established that both genetic variants and gene expression contribute to these observed population differences. Here we set out to evaluate to what extent population differences may be explained by an epigenetic factor, namely microRNA (miRNA) expression. We characterized genome-wide miRNA expression in unrelated 54 CEU (Utah residents with northern and western European ancestry) and 55 YRI (African from Ibadan, Nigeria) HapMap lymphoblastoid cell lines (LCLs) using the Exiqon miRCURYTM LNA arrays and evaluated baseline mRNA expression using Affymetrix GeneChip® Human Exon 1.0 ST array. Forty-one miRNA expression traits were found to differ significantly between these 2 ethnic groups (pBonferroni corrected<0.05). The potential biological function of these observed differentially expressed miRNAs was explored by examining their relationship with mRNA expression and cellular sensitivity to carboplatin, daunorubicin and cytaribin. After multiple testing adjustment (false discovery rate (FDR)<0.1), we identified 25 and 32 of the 41 differentially expressed miRNAs that were significantly correlated with 530 and 729 mRNA expression levels in the CEU and YRI samples, respectively. Among them, six differentially expressed miRNAs showed significant levels of correlation with 6 differentially expressed mRNAs (FDR<0.1). A substantial proportion (63%) of these miRNAs correlated with cellular sensitivity to chemotherapeutic agents (FDR<0.05). Lastly, upon performing a genome-wide association study between SNPs and miRNA expression, we identified a large number of SNPs showing high levels of population differentiation that affected the expression of these differentially expressed miRNAs, suggesting the role of genetic variants in mediating the observed population differences. In conclusion, the differentially expressed miRNAs we catalog in this study may explain observed population differences in mRNA expression and cellular sensitivity to chemotherapeutic drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2011-4731
Abstract 4734: Modeling acquired resistance to MAPK pathway inhibitors in KRAS mutant tumors
Authors: Liu Bonnie , O'Brien Carol , Song Kyung , Spoerke Jill , Moffat John , Seshagiri Somasekar , Friedman Lori S. , Belvin Marcia , Lackner Mark , Hatzivassiliou Georgia
Published: June 2012

Background: Acquired resistance to antitumor therapies underlies one of the challenges in effectively treating cancer. Understanding the mechanisms of acquired drug resistance may lead to elucidation of redundant oncogenic pathways and the development of more effective therapies. The RAS/RAF/MEK pathway is active in over 30% of human tumors, often due to mutation in BRAF or RAS family members. Several MEK inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in various stages of clinical development. These inhibitors are allosteric and act by binding to a hydrophobic pocket adjacent to the ATP binding pocket to inhibit MEK kinase activity. Preclinical studies using randomly mutagenized MEK cDNAs have identified mutations in MEK that upon transfection in MEK sensitive lines can render them resistant to MEK inhibitors. The strongest effects were observed with mutations affecting the allosteric binding pocket, thus blocking inhibitor binding. Results: To model acquired resistance to MEK inhibitors 3 independent KRAS mutant tumor cell lines (breast MDA-MB-231, colon HCT116 and LoVo) were grown in the presence of increasing concentrations of the selective MEK inhibitor PD0325901 and pools of MEK resistant cells were characterized versus parental cells. MEK resistant cells were still dependent on the MAPK pathway and sensitive to treatment with inhibitors against the downstream kinase ERK. In all three lines, MEK resistance was associated with acquisition of mutations in the MEK allosteric binding pocket, resulting in lack of signaling inhibition and suggesting that such resistance can arise relatively rapidly and in distinct tumor types. To compare mechanisms of acquired resistance to ERK inhibitors, we also generated HCT116 cells resistant to a selective ERK inhibitor. ERK resistant lines remained sensitive to MEK inhibitors and dependent on the MAPK pathway. Conclusions: Acquired resistance to MEK inhibitors in KRAS mutant cells arises through mutations in MEK that block inhibitor binding so that the inhibitor is no longer effective. In this case, tumors remain dependent on the MAPK pathway and can be targeted by inhibitors that act downstream of mutated target. These studies provide the rationale for future screening of patients with acquired MEK inhibitor resistance for mutations in MEK1 and MEK2. Patients whose resistance is driven through MEK mutations may benefit from treatment with an ERK inhibitor. Combination of MEK and ERK inhibitors may potentially prevent or delay the onset of resistance in MAPK-addicted tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2011-4734
Abstract 474: Tumor CD57+ immune cell infiltration is a strong independent prognostic factor in stage II-III colorectal cancer
Authors: Boige Valerie , Locher Clara , Svreck Magali , Auperin Anne , Chaput Nathalie
Published: June 2012

Background: CD3+ CD45RO+ tumor infiltrating lymphocytes (TIL) density within colorectal cancer (CRC) has been shown to be a better predictor of outcome than UICC TNM. Conversely, Laghi and coll. showed that CD3+ TIL density was not a prognostic factor in stage III CRC. The aim of our study was to evaluate the prognostic role of immune infiltrating tumor cells in stage II and III CRC patients using a tissue microarray (TMA) analysis.

Methods: A tissu-array was constructed using 202 stage II and III resected CRC (154 CC, 48 rectal cancers) and their matched non tumor tissue (NT). Tumors exhibiting deficient mismatch repair phenotype have been excluded. On the basis of previous data on the prognostic role of immune response in CRC, the following stainings were quantified in tumor (TT) and NT: CD3, CD68, CD57, CD8 and CD4 in order to quantify macrophage, lymphocyte cell infiltration as effectors of IFN-γ production, CXCL9/MIG, PPAR γ, CXCL10/IP-10, INDO, CXCL13/BCA-1. For prognostic analysis, immunohistochemistry (IHC) data was dichotomized at the median value.

Results: All proteins (except CXCL10/IP-10, INDO for which no reliable IHC data could be obtained) were significantly underexpressed in TT as compared to NT (p<0.0001, paired t-test). CD57 expression was significantly correlated with both CD3 and CD8 expression in TT (Spearman rank correlation coefficients equal to 0.41 (p<0.001) and 0.22 (p=0.002) respectively). After the exclusion of patients who received preoperative irradiation for rectal cancer (n=38), and after adjustment for tumor stage, adjuvant chemotherapy and tumor site in Cox models, CD3 and CD57 tumor staining were associated to recurrence-free survival (RFS) (HR=0.48 and 0.33 and p-value=0.02 and 0.003 respectively) whereas none of the NT staining was found to be prognostic. When CD3 and CD57 tumor staining were included together in the Cox model, only CD57 remained significantly associated with RFS (HR=0.37, p=0.015), especially in stage III CRC but without statistically significant interaction between stage and CD57.

Conclusion: Tumor CD57+ immune cell infiltration is associated with prolonged recurrence-free survival in stage II and III colorectal cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2011-474
Abstract 4745: TLR8 agonist and Doxil chemotherapy potently activate human antitumor immune response in a human immune system mouse model
Authors: Facciabene Andrea , Malykhin Alexander , Mathews Maura , Fang Chungyun , Dietsch Greg , Hershberg Rob , Coukos George
Published: June 2012

Because of differences between mouse and human immune systems, many of the effects of immunomodulatory drugs cannot be fully studied in syngeneic mouse models. We thus generated a novel tumor-bearing mouse model with human immune system (HIS) to study interactions between chemotherapy and immune modulatory therapy. We tested the individual effects and the interactions between doxorubicin, a drug which induces immunogenic tumor cell death and activates antigen-presenting cells, and VTX-2337, a novel small-molecule TLR8 agonist, which induces potent activation and type 1 polarization of human myeloid DCs but is inactive on murine leukocytes. Nod/SCID/ILRγc knock out (NSG) mice were inoculated with human CD34+ cord blood cells from HLA-A2+ human donors; transplanted s.c. with human HLA-A2+ OVCAR5 ovarian cancer tumors; and treated with pegylated liposomal doxorubicin (Doxil); VTX-2337; or a combination. NSG-HIS mice exhibited a full human hematopoietic system, including human monocytes, macrophages and plasmacytoid and myeloid DCs as well as T and B cell subsets. In NSG-HIS mice, VTX-2337 induced dose-dependent activation of human Monocytes CD14+ and CD11c+ cells with upregulation of CD80, CD86, CD83, CD40 and MHCII within 6 hrs. Transient, dose-dependent upregulation of human Th1 cytokines but also IL-10 was observed in the plasma of treated mice, reaching peaks within 6 hrs and subsiding within 24 hrs. Doxil induced mild activation of CD11c+ DCs and mild upregulation of Th1 cytokines. The combination of two drugs induced potent activation of CD11c+ DCs and marked increase in Th1 cytokines with significant decrease of IL-10. HLA-A2+ OVCAR5 tumors were successfully engrafted, exhibiting infiltration by human leukocytes. VTX-2337 and Doxil treatment independently induced tumor-infiltrating lymphocytes and restricted growth of human ovarian tumor xenografts in a dose-dependent manner, while the combination of the two drugs induced the highest frequency of Leukocytes (hCD45) and potently restricted growth of ovarian tumors. Combined activation of innate and adaptive immunity by VTX-2337 and Doxil, as well sensitization of tumor cells by Doxil to adaptive and innate immune effector mechanisms was at the basis of the observed interactions suppressing tumor growth. We conclude that the NSG-HIS provided a suitable tool to establish potent interactions between TLR8 agonist and Doxil chemotherapy, which warrant clinical testing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4745. doi:10.1158/1538-7445.AM2011-4745
Abstract 4748: Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation
Authors: Yamaguchi Hideki , Fukami Kiyoko , Sakai Ryuichi
Published: June 2012

Degradation of extracellular matrix (ECM) that is present in the basement membrane and tumor stroma is essential for local invasion and formation of metastatic sites by malignant cancer cells. Invadopodia are ECM-degrading protrusions formed by a variety of invasive cancer cell types and are thought to function in cancer invasion and metastasis. Many components of invadopodia, such as proteins involved in actin polymerization, cell signaling, and ECM degradation, have been reported to date. However, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that produce D-3 phosphoinositides. PI3Ks mediate the signal transduction of extracellular stimuli and regulate diverse cellular events. Uncontrolled activation of the PI3K signaling pathway leads to several pathological phenomena, including tumorigenesis and tumor malignancy. We investigated the role of PI3K signaling during invadopodia formation. We found that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix was blocked by treatment with PI3K inhibitors or sequestration of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. Mammals have 8 PI3K enzymes that are subdivided into three general classes (I-III) on the basis of their domain structures and substrate specificities. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α is selectively involved in invadopodia formation. p110α is encoded by the PIK3CA gene, which is one of the most frequently amplified and mutated genes identified in human cancers. Clinical studies revealed that activating mutations of PIK3CA are associated with the development of invasive and metastatic phenotypes and poor patient prognosis. The expression of p110α with the activating mutations promoted invadopodia-mediated invasive activity. Furthermore, knocking down PDK1 and Akt, the downstream effectors of PI3K signaling, suppressed invadopodia formation induced by the p110α mutants. These data suggest that the PI3K signaling pathway via p110α regulates invadopodia-mediated invasion of breast cancer cells. Targeting p110α may be a potential therapeutic approach for controlling invasion of malignant cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2011-4748
Abstract 4754: Targeting of Ewing sarcoma by CAR-reengineered GD2-specific T cells
Authors: Kailayangiri Sareetha , Altvater Bianca , Meltzer Jutta , Leuchte Katharina , Hotfilder Marc , Juergens Heribert , Rossig Claudia
Published: June 2012

Primary metastatic Ewing sarcoma involving the bone and/or bone marrow has remained largely incurable. Relapses after intensive multimodal treatment regimens are attributed to a rare subpopulation of residual cells capable of reinitiating tumor growth. Cellular immunotherapy with chimeric antigen receptor (CAR) transduced T cells is a promising strategy to eradicate minimal residual disease and prevent relapse. Based on the observation that Ewing sarcoma cells express the ganglioside antigen GD2, we have explored the capacity of T cells transduced with GD2-specific CARs to functionally interact with Ewing sarcoma cells.

Surface GD2 expression was detected on 10 of 11 Ewing sarcoma cell lines by flow cytometry analysis, and on 11 of 12 primary Ewing tumor samples by immunofluorescence staining. To obtain GD2-specific effector T cells, CD3/CD28 stimulated T cells from five healthy donors were retrovirally transduced with the second generation GD2-specific CAR 14.G2aCD28ζ, resulting in CAR surface expression in 83.8±2.7% (81.1-87.8%) of the cells. Ewing sarcoma cell lines expressing various levels of GD2 were used as target cell lines for 14.G2aCD28ζ-mediated cytolysis in a 51Cr release assay. Both GD2hi VH-64 and GD2intermediate Cado-ES-1 cells were efficiently lysed by gene-modified T cells (36.6±7.7% and 25.3%±8.2 lysis at a 40:1 effector-to-target cell ratio) in the absence of background cytolysis of GD2-negative tumor cells and by non-transduced T cells. Moreover, efficient and antigen-specific granzyme B secretion was found after coincubation of various Ewing sarcoma cell lines and a primary Ewing sarcoma cell culture with CAR gene-modified T cells. 14.G2aCD28ζ T cells further eradicated Ewing sarcoma cells in a serum-free spheroid model for anchorage-independent micrometastatic tumor growth. Functional activation of CAR-expressing T cells in response to GD2 positive Ewing sarcoma cells was further demonstrated by significant secretion of IFN-γ and TNFα and significant proliferative responses to various cell lines expressing low, intermediate or high levels of GD2.

Thus, GD2 is a promising target antigen for the redirected antitumor activity of T cells not only in neuroblastoma and melanoma, but also in Ewing sarcoma. To address the preclinical in vivo efficacy of 14.G2aCD28ζ T cells, we have now established an in vivo model of disseminated disease by intravenous transfer of Ewing sarcoma cells to sublethally irradiated NOD/scid mice. Magnetic resonance imaging allows to visualize metastatic tumor manifestations in xenotransplanted mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4754. doi:10.1158/1538-7445.AM2011-4754
Abstract 4760: Molecular classification and risk-stratification in medulloblastoma using an integrative genomic approach
Authors: Cho Yoon-Jae , Tamayo Pablo , Tsherniak Aviad , Meyerson Matthew , Mesirov Jill P. , Pomeroy Scott L.
Published: June 2012

Purpose. Medulloblastomas are heterogeneous tumors that collectively represent the most common malignant brain tumor in children. To understand the molecular characteristics underlying their heterogeneity and to identify whether such characteristics represent risk factors for patients with this disease, we performed an integrated genomic analysis of a large series of primary tumors.

Patients and Methods. We profiled the mRNA transcriptome of 194 medulloblastomas and performed high-density SNP array and miRNA analysis on 115 and 98 of these, respectively. Unsupervised clustering analysis of mRNA expression data was used to identify molecular subgroups of medulloblastoma and DNA copy number, miRNA profiles, and clinical outcomes were analyzed for each. We then developed a Bayesian cumulative log odds model for predicting outcome, starting with ‘evidence’ provided by clinical features (metastasis and histology), then incrementally adding genomic evidence representing disease-subtype independent and dependent features. We then validated our results on independent test cohorts.

Results. We identify 6 molecular subgroups of medulloblastoma, each with a unique combination of numerical and structural chromosomal aberrations that globally influence mRNA and miRNA expression. We reveal the relative contribution of each subgroup to clinical outcome as a whole and show that a previously unidentified molecular subgroup, characterized genetically by c-MYC copy number gains and transcriptionally by enrichment of photoreceptor pathways and increased miR-183∼96∼182 expression, is associated with significantly lower rates of event free and overall survival. Moreover, we show that integration of high-level clinical and genomic risk factors, including molecular subtype, can yield more comprehensive, accurate and biologically interpretable prediction models for outcome in medulloblastoma. We introduce a novel Bayesian nomogram indicating the amount of evidence that each feature contributes on a patient-by-patient basis.

Conclusion. Our results detail the complex genomic heterogeneity of medulloblastomas, identify a previously unrecognized molecular subgroup with particularly poor clinical outcome and propose a Bayesian outcomes prediction model that outperforms the current clinical classification schema and previously published molecular markers proposed for medulloblastoma risk stratification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4760. doi:10.1158/1538-7445.AM2011-4760
Abstract 4762: Genistein and Indole 3 carbinol induced apoptosis in endometrial cancer cell through DR5 mediated pathway
Authors: Cho Chi-Heum , Shin So-Jin , Kwon Sang-Hoon , Cha Soon-Do , Kim Yong-Man
Published: June 2012

Objective: To address the growth inhibitory effects of Genistein and Indole 3 carbinol in endometrial cancer cell growth and evaluate its effect on apoptosis using ishikawa endometrial cancer cell line.

Methods: Human endometrial cancer cells ishikawa were cultured and treated with various concentrations of Genistein and Indole 3 carbinol alone and in combination. Cell proliferation and growth arrest were evaluated by MTS assay and FACS analysis, respectively. The apoptosis induced was analyzed by DNA fragmentation and western blotting.

Results: Combination of Genistein and Indole 3 carbinol significantly inhibited the proliferation of ishikawa cells in dose dependent manner. Cell cycle analysis indicated the increased proportion of cell arrest in sub G1 phase. DNA fragmentation, up regulation of DR5, down regulation of c-Flip, cleavage of caspase-8 and PARP revealed apoptotic progression.

Conclusion: Genistein and Indole 3 carbinol synergistically inhibited the growth of endometrial cancer cell. Significant induction of apoptosis is associated with DR5 regulated death dependent pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4762. doi:10.1158/1538-7445.AM2011-4762
Abstract 4765: Investigation of omega-3 docosahexaenoic acid (DHA)-induced apoptosis in human triple negative breast cancer
Authors: Xiong Ailian , Yu Weiping , Sanders Bob G. , Kline Kimberly
Published: June 2012

Omega-3 fatty acid enriched diets especially diets rich in docosahexaenoic acid (DHA) have long been associated with a lower incidence of a variety of cancers. Both in vivo and in vitro studies have indicated that DHA exerts anticancer actions via inducing tumor cells to undergo programmed cell death or decreasing cell proliferation. In this study, we investigated the anticancer actions of DHA in human triple negative breast cancer (TNBC) cells in vitro. Data indicate that DHA induces apoptosis in MDA-MB-231 and BT-20 human p53 mutant TNBC cells by activating both intrinsic and extrinsic death-mediating pathways. Activation of apoptosis is associated with induction of endoplasmic reticulum (ER) stress, and increased levels of phosphorylated JNK (pJNK), and CHOP and DR5 protein levels; as well as, reduced levels of pIκB and NF-κB downstream mediators: c-FLIP, Survivin and Bcl-2. DHA induces ROS generation and antioxidants, RRR-alpha tocopherol (αT) and N-acetyl-Cysteine (NAC) block DHA-induced apoptosis, ER stress and upregulation of pJNK/CHOP/DR5. Taken together, our data demonstrate that DHA induces apoptosis in p53 mutant TNBC via activation of ROS mediated ER stress and suppression of NF-kB mediated anti-apoptotic factors. Since p53 mutant TNBCs are associated with poor prognosis and limited treatment options, these studies suggest DHA may be a successful adjuvant treatment strategy. Research is supported by the Clayton Foundation for Research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2011-4765
Abstract 4768: Receptor interacting protein (RIP) and Sirtuin-3 (SIRT3) on opposite sides of anoikis resistance and promotes tumorigenesis
Authors: Kamarajan Pachiyappan , Alhazzazi Turki , D'Silva Nisha , Verdin Eric , Kapila Yvonne
Published: June 2012

Anoikis-apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts is disregulated in many chronic debilitating and fatal diseases. Regulating crosstalk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like autoimmune diseases and cancer. Previously, we showed that anoikis activates a CD95/Fas-mediated signaling pathway regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/FAK-mediated survival pathways in a variety of cell systems. Since sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is known to regulate longevity, cell survival, metabolism, and tumorigenesis, we hypothesized that SIRT3, might engage in crosstalk with Fas/RIP/integrin/FAK survival-death pathways. Specifically, we examined the roles of RIP and SIRT3 in oral squamous cell carcinoma (OSCC) anoikis resistance and tumorigenesis. Using immunohistochemical staining, immunoblotting, human tissue micro arrays, and overexpression and suppression approaches we examined the roles of RIP and SIRT3 in this context. RIP and SIRT3 have an opposite expression profile in OSCC cells and tissues. Stable suppression of RIP by RIP-targeted shRNA enhances SIRT3 levels, whereas, stable suppression of SIRT3 by SIRT3-targeted shRNA does not impact RIP levels in OSCC cells. Forced expression of RIP in RIP null cells downregulates SIRT3. As OSCC cells become anoikis-resitant they form multicellular aggregates in suspension conditions and their expression of SIRT3 increases as their expression of RIP decreases. Anoikis-resistant cells with high SIRT3 and low RIP levels induce an increased tumor burden and incidence in mice unlike their adherent OSCC cell counterparts, which express low SIRT3 and high RIP levels. OSCC cells with SIRT3 suppression fails to form multicellular aggregates in suspension conditions. Taken together, these data demonstrate that RIP is an upstream negative regulator of SIRT3 in anoikis resistance and anoikis resistance contributes to a more aggressive phenotype in OSCC development and progression (Supported by NIH grant R01DE014429 and NIH grant 2R56DE014429 to YLK).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2011-4768
Abstract 4778: Resveratrol suppresses constitutive activation of AKT via generation of reactive oxygen species and induces apoptosis in diffuse large B cell lymphoma cell lines
Authors: Jehan Zeenath , Hussain Azhar R. , Ahmed Maqbool , Ahmed Saeeda O. , Al-Dayel Fouad , Bavi Prashant , Uddin Shahab , Al-Kuraya Khawla S.
Published: June 2012

We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in the pathogenesis of diffuse large B cell lymphoma (DLBCL). In an attempt to identify newer agents that can be utilized to target these deregulated pathways, we found that Resveratrol (trans-3,4′, 5-trihydroxystilbene) is a naturally occurring polyphenolic compound that has an antioxidant and chemopreventive effects on various cancer cells. In the current investigation, we therefore investigated the role of resveratrol-mediated chemopreventive effect on various DLBCL cell lines. Resveratrol caused a dose dependent inhibition of cell viability in DBCL cell lines. To see whether this inhibition was due to cell cycle arrest or apoptosis, we found that resveratrol induced apoptosis in all DLBCL cell lines. We next investigated the action of resveratrol on PI3-kinase/AKT pathway and found that resveratrol treatment resulted in inhibition of constitutively activated AKT and its downstream targets, p-FOXO1, p-GSK3 and p-Bad via generation of reactive oxygen species (ROS). Inactivation of Bad led to conformational changes in Bax protein causing loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. This led to activation of caspase-9, caspase-3, and poly-(ADP)-ribose polymerase cleavage leading to caspase-dependent apoptosis. In addition, resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; however, this up-regulation did not result in apoptosis. Finally, co-treatment of DLBCL with sub-toxic doses of TRAIL and resveratrol induced efficient apoptosis. Altogether, these data suggest a novel function for resveratrol, acting as a suppressor of AKT/PKB pathway via generation of ROS in DLBCL cells, and raise the possibility that this agent may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway either alone or in combination with other chemotherapeutic agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4778. doi:10.1158/1538-7445.AM2011-4778
Abstract 4779: TNF-induced Cofilin 1 phosphorylation by LIMK in colorectal tumor cells
Authors: Ivanovska Jelena , Ettle Benjamin , Benderska Natalya , Chakilam Saritha , Gandesiri Muktheshwar , Ziesché Elisabeth , Hartmann Arndt , Agaimy Abbas , Fischer Thomas , Gohla Antje , Fabry Ben , Schneider-Stock Regine
Published: June 2012

Objectives: Recently, we have shown that the pro-inflammatory cytokine TNF induces apoptosis in HCT116 colorectal tumor cells. Among the first apoptotic signs is a drastic change in cell morphology through modulation of the cytoskeleton. In order to identify and to characterize phosphorylation events and kinase targets associated with early cytoskeletal reorganization after TNF-treatment, we performed a peptide array screen.

Methods: HCT116 tumor cells were cultured for 6h to 72 hours in either normal or TNF- conditioned medium, and radioactive labelled lysates were hybridized to a PepChip kinase full slide array for screening the phosphorylation status of over 400 proteins at over 1400 potential phosphorylation sites. For verification, cell lysates were collected and co-immunoprecipitated. The precipitated proteins were analyzed by western blotting. We verified the subcellular localization of proteins by co-immunofluorescence.

Results: Pepscan array and western blotting revealed that cofilin 1 is markedly phosphorylated on Serine 3 (pCofSer3) after 24 and 48 hours of TNF-treatment. A potential up-stream kinase of cofilin is LIMK1, a serine/threonine protein kinase known to be involved in actin cytoskeleton reorganization through phosphorylation and inactivation of cofilin 1. Interaction between LIMK/Cofilin1/pCofSer3 was verified by co-immunoprecipitation and co-immunofluorescence. F-actin, cofilin1 and LIMK colocalized in membrane ruffles and lamellipodia, consistent with a cofilin-dependent actin cytoskeletal remodelling after TNF treatment. The effects were reversed by ROCK 1 inhibition.

Conclusion: Our data show that TNF-treatment in HCT116 tumor cells induces the formation of a LIMK/Cofilin1/pCofSer3 protein complex that is localized in membrane ruffles and lamellipodia, suggesting that TNF-induced cytoskeletal alterations early during apoptosis are regulated through cofilin signalling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4779. doi:10.1158/1538-7445.AM2011-4779
Abstract 4816: Overexpression of thymosin β10 gene is not mediated by DNA methylation in non-small cell lung cancer
Authors: Lee Su Man , Lee Ji Yun , Kim Mi Jin , Yoon Ghil Suk , Park Jae Yong , Kim Dong Sun
Published: June 2012

Lung cancer is the leading cause of cancer-related deaths worldwide and is usually associated with a late diagnosis and a poor prognosis. Thymosin β10 (TMSB10) is monomeric actin sequestering protein to regulate actin cytoskeleton organization. The aberrant TMSB10 expression has been implicated in the pathogenesis of human cancers. However, its role in carcinogenesis is still controversial. To better understand the role of TMSB10 in lung tumorigenesis and its regulatory mechanism, we have examined the methylation status and expression of the TMSB10 gene in non-small cell lung cancers (NSCLCs) using methylation-specific PCR (MSP) and immunohistochemistry (IHC), respectively. MSP analysis showed that TMSB10 promoter was almost unmethylated in tumor tissues and became to be hypomethylated in 7 out of 139 (5.0%) NSCLCs. However, there is no significant association of TB10 demethylation with clinicopathologic features. IHC showed that TMSB10 protein was strongly expressed in the cytoplasm of malignant cells and its overexpression was detected in 15 of 30 (50.0%) the tumor tissues compared to normal tissues. Moreover, TMSB10 overexpression was frequently observed in sqaumous cell carcinomas compared to adenocarcinomas with border line significance (P = 0.072), but its overexpression was not associated with the methylation status of TMSB10 gene. Collectively, these results suggest that TMSB10 overexpression may be a frequent event in the progression of NSCLC, but DNA methylation may not involve in TMSB10 overexpression. However, further studies with a large number of cases are needed to confirm our findings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4816. doi:10.1158/1538-7445.AM2011-4816
Abstract 4819: Wolf-Hirschhorn syndrome candidate 1, a histone lysine methyltransferase, is involved in human carcinogenesis
Authors: Hamamoto Ryuji , Yoshimatsu Masanori , Toyokawa Gouji , Cho Hyun-Soo , Nakamura Yusuke
Published: June 2012

A number of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases like cancer are not well understood. Here, we demonstrate that Wolf-Hirschhorn syndrome candidate 1 (WHSC1) plays an important role in human carcinogenesis. Transcriptional levels of this gene are significantly elevated in various types of cancer including bladder and lung cancers. Immunohistochemical analysis using a number of clinical tissues confirmed significant up-regulation of WHSC1 expression in bladder and lung cancer cells at the protein level. Treatment of cancer cell lines with small interfering RNAs targeting WHSC1 significantly knocked down its expression and resulted in the suppression of proliferation. Moreover, knockdown of WHSC1 decreased the cell population of cancer cells at S phase and increased that at G2/M phase through the regulation of genes involved in the Wnt signaling pathway. Furthermore, we found WHSC1 to be interacted with some proteins related to the Wnt pathway including β-catenin. As expression levels of WHSC1 are significantly low in normal tissues, it may be feasible to develop the inhibitors targeting these enzymes as anti-tumor agents which have a minimal risk of adverse reaction.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4819. doi:10.1158/1538-7445.AM2011-4819
Abstract 4820: Whole methylome explorations of paired tumor and non-tumor clinical samples using HELP
Authors: Mullapudi Nandita , Shi Miao , Suzuki Masako , Spivack Simon
Published: June 2012

Introduction: Gene silencing by promoter methylation is common in carcinogenesis. In lung cancer, hypermethylation of specific gene promoters has been found to occur during the earliest histologic stages of the cancer. Thus far surveys of methylation in lung tumors have focused on specific gene panels (5 – 10 genes). A few large scale studies have reported selection of longer gene lists or focused on larger sets of CpG islands.

Methods: We have adapted an approach to survey genome-wide methylation events in tumor and adjacent non-tumor tissue from clinical lung resection samples using the HELP (HpaII tiny fragment Enrichment by Ligation mediated PCR) assay. This assay, based on the generation of restriction enzyme libraries generated by methylation sensitive (HpaII) and methylation insensitive (MspI) isoschizomers yields information on 1.2 million HpaII fragments throughout the genome. Each HpaII fragment corresponds to two CpG sites. The assay lends itself to low starting amounts of DNA (3 ug) and robust assessment of methylation status by comparing ratios of HpaII- generated- fragments to MspI- generated fragments co-hybridized to a Nimbelgen custom high-density microarray.

Summary of results: In a pilot set of three paired tumor and non-tumor tissue samples, we identified 1504 fragments (corresponding to two CpG sites each) that were hypermethylated in tumor versus non-tumor in all three pairs. Of these, 165 fragments were located within gene promoters. In the opposite direction, 2907 fragments were found to be hypermethylated in non-tumor versus tumor. Of these, 81 were located within gene promoters. We could identify both known and novel regions of the genome as well as specific gene-promoters that are hypermethylated in tumor versus non-tumor. We will discuss selected methylation events in further detail.

Conclusion: The HELP assay is a powerful approach to interrogate methylation status of individual HpaII fragments across the whole genome, offering a large sample size of 1.2 million HpaII fragments. We are able to discover individual methylation events common across different clinical specimens and are currently expanding our sample set, and planning correlations with transcriptome-wide studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4820. doi:10.1158/1538-7445.AM2011-4820