bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1266: Glioma biomarker discovery by mass spectrometry
Authors: Dekker Lennard , Kheirollahi Majid , Stockhammer Guenther , Luider Theo , Smitt Peter Sillevis
Published: August 2012

BACKGROUND Approximately 200.000 people are diagnosed with a primary brain tumor each year worldwide. The most common type of primary brain tumor (∼40%) are gliomas. Currently available prognostic markers require evaluation of tumor tissue, such as 1p and 19q choromosome co-deletion in oligodendroglial tumors, MGMT promoter methylation for glioblastomas (GBMs), and IDH mutations in several glioma subtypes. Serum biomarkers that correlate with tumor status have prognostic and predictive value and could significantly improve the care for glioma patients. In addition biomarkers could provide unique information related to longitudinal response to therapy. The only clinically validated serum marker of prognosis and disease status in high grade gliomas is serum YKL-40, a chitinase homolog also called human cartilage glycoprotein 39 or chitinase 3-like 1. DESIGN/METHODS For the discovery phase we used a set of 31 cerebrospinal fluid samples. This sample set included 5 CSF samples of patients with a GBM and 26 controls. These samples were digested by trypsin and subsequently measured by LCMS-Orbitrap. The acquired data were analyzed using a database search and a subsequent analyses of the spectral counts by Scaffold. The spectral counts for each individual identified protein are used as a semi quantitative measure for protein abundance. RESULTS / CONCLUSIONS From the total list of 771 identified proteins, candidate biomarker proteins were selected that showed a significant difference between the control and GBM (p<0.01) and were at least 3 fold higher in spectral count than in the control group. Abundant serum proteins were removed to avoid inclusion of proteins that relate to the disruption of the blood-CSF barrier. Eventually, a list of eleven proteins was obtained that included 14-3-3 protein gamma, 72 kDa type IV collagenase, Alpha-enolase, Chitinase-3-like protein 1 (YKL-40), Fetuin-B, Glial fibrillary acidic protein, Insulin-like growth factor II, Metalloproteinase inhibitor 1, Serum paraoxonase/arylesterase 1, SPARC, IGF binding protein 2 and Metalloproteinase inhibitor 2. Many of the identified candidate markers have been previously reported in literature in relation to glioma including the clinically validated serum marker for glioma YKL-40. We are now planning a validation of these markers in a set of serum samples of glioma patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1266. doi:1538-7445.AM2012-1266
Abstract 1269: Activation of diverse signaling pathways in pancreatic cancer revealed by phosphoproteomics
Authors: Kim Min-Sik , Chaerkady Raghothama , Wu Xinyan , O'meally Robert , Kuppireddy Sarada Vineela , Zhong Jun , Jacob Harrys KC , Marimuthu Arivusudar , Kashyap Manoj K. , Cole Robert , Iacobuzio-Donahue Christine , Maitra Anirban , Pandey Akhilesh
Published: August 2012

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. A number of genes that have somatic mutations including CDKN2A, SMAD4, TP53 and KRAS have been linked to pancreatic cancer. Recently, a comprehensive genomic analysis was performed in a panel of 14 primary pancreatic cancer cell lines and 10 xenografts, in which ∼1,500 somatic alterations were identified and a core set of 12 activated signaling pathways were defined. We decided to use a phosphoproteomics approach to identify tyrosine kinase signaling pathways that could serve as attractive therapeutic targets. Based on immunoblotting with anti-phosphotyrosine antibodies, we observed a heterogeneous pattern of tyrosine phosphorylation in the 14 primary pancreatic cancer cell lines whose genomes have been sequenced previously, which indicated that different tyrosine kinases were likely activated in different cells. To identify these kinases, phosphotyrosine-containing peptides were enriched by immunoprecipitation by anti-phosphotyrosine antibodies and analyzed on a high resolution Fourier transform mass spectrometer coupled to nanoflow reversed phase liquid chromatography. W employed the SILAC methodology to quantitate tyrosine phosphorylated peptides across the panel of cell lines. Our preliminary data indicates that the approach used in this study is suitable for global profiling of tyrosine kinase pathways that are abnormally activated in cancers. The differential alteration of tyrosine kinases was confirmed from the accurate quantitative tyrosine phosphoproteomic analysis. For example, HPNE, a normal pancreatic cell line showed the lowest level of phosphotyrosine levels, while each pancreatic cancer cell line could be differentiated based on its own set of activated tyrosine kinases. Interestingly, some of tyrosine kinases were activated in a small number of cell lines (e.g. AXL, SYK, DDR2, RON and RET), while other tyrosine kinases were found to be activated more broadly (e.g. YES1, TYK2, EPHA2 and EPHB4). Overall, we conclude that these aberrantly activated tyrosine kinases could be targeted to develop newer therapeutic regimens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1269. doi:1538-7445.AM2012-1269
Abstract 1272: MALDI imaging and next generation sequencing for dissecting prostate cancer heterogeneity
Authors: Stahl Phillip , Wurlitzer Markus , Schlüter Hartmut , Thiele Herbert , Becker Michael , Simon Ronald , Weischenfeldt Joachim , Korbel Jan , Minner Sarah , Sauter Guido
Published: August 2012

Prostate cancer is often heterogeneous with multiple tumor foci within the same prostate. MALDI (Matrix-Assisted Laser Desorption/Ionization) mass spectrometry imaging (MSI) allows for a comprehensive profiling of the molecular phenotype on the protein, peptide, lipid, and metabolite level across entire tissue slides without prior enrichment of tumor cells. This study aims in a MALDI-based discrimination of cancer areas that are heterogeneous with respect to morphology and molecular features. We established a method for cryopreservation of entire prostates from cancer patients, and build tissue microarrays from individual cancerous prostates representing >30 different areas of the primary tumor as well as multiple lymph node metastases. We analyzed these arrays for ERG fusion by FISH and for peptide signatures by MALDI MSI. In addition, 2 selected tumor areas and 4 different lymph node metastases from one prostate were subjected to paired end next generation sequencing. MALDI identified 5 masses that were specific for epithelial cells. One particular mass was strongly related to presence of ERG fusion. In one prostate, we identified another mass that was present only in 2/35 tissue spots from the primary tumor as well as in 4/10 lymph nodes. Deep sequencing of different tumor areas of this case identified distinct patterns of structural genetic alterations. A cluster analysis showed that all samples were clonally related, and identified the area inside the primary tumor that gave rise to the metastases. In conclusion, these data suggest that MALDI imaging and next generation sequencing allow for identification of genetic and proteome patterns that correspond to metastatic spread of prostate cancer cells. A combination of both techniques is suited to dissect the genetic background of distinct MALDI signatures.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1272. doi:1538-7445.AM2012-1272
Abstract 1291: Tumor growth inhibitory effect in EIF4B silenced in vitro and in vivo lung cancer models
Authors: Shin Jung-Young , Zhang Xiang-Hua , Kim Jeong-Oh , Oh Ji-Eun , Chae Hiun Suk , Joe Young Ae , Kang Jin-Hyoung
Published: August 2012

Backgraound The eIF4B, a key molecule for initiation of protein translation, play an important role in cell growth, proliferation and survival. We investigated that eIF4B is associated with the molecular mechanism of de novo resistance against EGFR TKIs and its role on cell proliferation and survival in NSCLC cells. Method Among five NSCLC cell lines harboring EGFR and K-RAS wild type, PC14 cells showing the highest IC50, were selected for our experiment. In eIF4B silenced PC14 cells by shRNA, we evaluated cell proliferation by direct cell counts and SRB assay and cell cycle distribution using flow cytometry. The expression of signal molecules, transcription factors and apoptosis associated proteins was measured by Western blot. In vivo tumor model established by subcutaneous injection of eIF4B silenced PC14 cells, the retardation of tumor growth was measured. Results In PC14 cells transformed with eIF4B shRNA, we observed the protein expression of eIF4B was reduced (>70% at day 5). The proliferation of eIF4B silenced cells was greatly inhibited both in vitro and in vivo tumor model. Between eIF4B silenced cells and control cells, there was no significant difference in anti-proliferative activity of several target inhibitors (enzastaurin, gefitinib, LY294002, vandetanib) against EGFR-PI3K-AKT signaling molecules. The expression of proteins involved in cell survival (pEGFR, pAKT and mTOR), transcription factors (pNFkB and TS) and anti-apoptotic protein (bcl-2), was decreased and apoptosis associated proteins (phospho-p53 and active caspase-3) were increased. The G0/G1 cell fraction in eIF4B silenced cells was reduced (47.3% vs. 55.1% in control cells) and accumulation of apoptotic cells was observed (12.7% vs. 1.5% in control cells; shown as sub G1 population). Conclusion Our results demonstrated that eIF4B silencing resulted in a marked decrease of several anti-apoptotic and pro-proliferative proteins and eIF4B was required for cell proliferation and survival by regulating cell cycle and cell signaling molecules.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1291. doi:1538-7445.AM2012-1291
Abstract 1294: Suppression of uPA in chemo-sensitization and p53 regulation in pancreatic cancer cells
Authors: Gorantla Bharathi , Asuthkar Swapna , Estes Norman , Rao Jasti S. , Gondi Christopher S.
Published: August 2012

Despite new knowledge of the molecular profile of pancreatic cancer and its precursor lesions, survival rates have changed very little over the last 40 years. The ability to predict which patients would benefit most from surgical intervention and/or chemotherapy would be a great clinical asset. Researchers have demonstrated that uPA is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients. It is known that uPA is involved in ECM degradation and has been correlated with malignant transformation of cancer cells. Emerging data have suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor called cancer stem cells (CSCs) or cancer initiating cells. Under in vitro cell culture conditions, these cells possess sphere-forming ability, which is known to be one of the properties of CSCs. Interestingly, the significance of this sphere formation ability still remains unclear. Recent studies have demonstrated that these sphere-forming cells acquired chemoresistance to cisplatin. In the present study, as observed by FACS analysis, we show that a sub-population of MIA PaCa-2 and PANC-1 pancreatic cancer cells possess cancer stem cell properties. RNAi-mediated suppression of uPA in these cells retarded their tumor forming ability in a nude mouse model. We also observed that uPA-suppressed MIA PaCa-2 and PANC-1 cells, after supplementation with uPA protein regained their cancer stem cell phenotype and showed increased expression of Lhx-2. To determine whether these sub-population cells possess any chemoresistance, we treated these cells with varying concentrations of gemcitabine (0-1000 nM) and observed that these cells were resistant to gemcitabine even at the 1000 nM concentration. Further suppression of uPA using siRNA in these cells sensitized these cells to gemcitabine. In addition, to determine whether uPA has any transcriptional role, we examined whether uPA interacted with any known transcription factors we used a TF-protein array. We observed that uPA interacted with Hoxa-5 very strongly. To further determine whether the interaction of uPA with Hoxa-5 invokes any regulatory response, we used a p53 promoter known to possess Hoxa-5 binding regions to drive a Luciferace gene, both with and without the presence of uPA. We observed that uPA retarded p53 promoter activity. Finally, nude mice implanted with MIA PaCa-2 cancer stem cells were susceptible to gemcitabine treatment after uPA downregulation. Taken together, these results indicate that targeting uPA can sensitize chemoresistant pancreatic cells to gemcitabine.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1294. doi:1538-7445.AM2012-1294
Abstract 1298: TWIST1 is expressed by cells with fibroblastic phenotype in colon carcinoma
Authors: Irene Gomez , Paloma Martin , Mercedes Herrera , Esther Martinez , Alberto Herrera , Vanesa garcia , javier Silva , Gemma Dominguez , Cristina Peña , Felix Bonilla , Miguel Garcia Jose
Published: August 2012

Twist1 is a highly conserved transcription factor that belongs to the family of basic helix-loop-helix proteins, a family of proteins with well-described functions in cell growth and differentiation, in both vertebrates and invertebrates. TWIST1 is involved in epithelial-to-mesenchymal transition (EMT), especially in invasiveness processes. Recently we have demonstrated that the expression levels of TWIST1 in colon tumor tissues can be considered as a poor prognostic marker (Gomez I et al, Plos One 28; e18023). Following in this research line we are trying to identify the specific cells expressing TWIST1 in the tumor mass, epithelial cells or fibroblastic phenotype cells, as is expected for cells in EMT. With this purpose we have isolated epithelial cells from 52 colon tumor tissues (based on the expression of EPCAM) and fibroblasts from 18 of them (based in the expression of a fibroblastic marker, Miltenyi Biotec), after mechanical cell dissociation of the different cells populations present in the tumor mass. TWIST1 expression profile was also tested in 13 tumor colon-derived cells and in the immortalized normal fibroblasts, IMR90. First we found that while 51.9 % of tumors samples expressed TWIST1, this only occurred in 1 out of 13 colon carcinoma cell lines tested, besides the IMR90 fibroblasts. Moreover, expression of TWIST1 was never detected in the epithelial fractions but was detected in 17 out of 34 non-epithelial fractions and in 10 out of the 18 samples where fibroblasts were specifically isolated. The absence of epithelial markers, Ber-EP4 and CD326 according with our isolation system, and CK18 and E-cadherin, verified by subsequent western blot, in the cells expressing TWIST1, suggests a non-epithelial origin of this cell population. Additionally, IMR90 fibroblasts co-cultured with LS174T or SW620 colon carcinoma cells showed increased TWIST1 expression (p = 0.002; p = 0.009 respectively), which was associated to up-regulation of the myofibroblast marker α-smooth muscle actin (α-SMA). Finally, there was a correlation between TWIST1 mRNA levels and tumor desmoplasia degree among those TWIST1 positive cases. We conclude that TWIST1 expression in colon adenocarcinomas corresponds to carcinoma-associated fibroblasts and could be implicated in their activation process.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1298. doi:1538-7445.AM2012-1298
Abstract 1301: Post translationally modified EVI1 regulates ΔNp63 to delay cell cycle progression and cell proliferation via the cyclin dependent kinase p21
Authors: Nayak Kasturi , Chakraborty Soumen
Published: August 2012

Several lines of evidence suggest that specific transcriptional events are involved in cell cycle, proliferation and differentiation processes; however, their deregulation by proto-oncogenes are involved in the development of leukemia and tumors. One such proto-oncogene is Ecotropic Viral Integration site I (EVI1) which can differentially effect cell cycle progression and proliferation in cell types of different origin. The proto-oncogene EVI1 which localizes in chromosome 3 (3q26.2) is attracting considerable attention in the recent years because of its reported involvement in late stage chronic myeloid leukemia (CML), high risk myelodysplastic syndrome (both MDS and MDS-AML) and also in de-novo acute myeloid leukemias (AML). Studies also indicate that EVI1 is overexpressed in a subset of human colon cancers, ovarian cancers and that EVI1 might induce disease progression and/or sensitivity to chemotherapy. In this report we provide evidence of a pathway through which EVI1 inhibits cell cycle progression and cell proliferation in HT-29 colon carcinoma cells which was reported earlier to express high levels of the proto-oncogene EVI1. Our bio-informatics analysis and quantified chromatin immunoprecipitation data shows that EVI1 very strongly binds to ΔNp63 promoter element. Deletion mutant analysis revealed that EVI1 binds to ΔNp63 promoter element through its distal zinc finger domain. RQ-PCR and luciferase assay revealed that the binding of EVI1 to ΔNp63 down regulates its expression by ∼4.5 fold and that down regulation of ΔNp63 induces the expression of p21 in HT-29 cells which eventually delay cell cycle progression at G0/G1 phase. Concomitant knocking down of EVI1 with a specific siRNA or transfecting ΔNp63 in EVI1+ cells significantly rescued this phenotype, indicating the growth defect induced by ΔNp63 deficiency to be, at least in part, attributable to EVI1 function. We have reported earlier that EVI1 is post-translationally modified and the modified forms of EVI1 divergently regulate the target genes. Post-translational modification of proteins is a hallmark of signal transduction and it allows existing proteins to react rapidly to extra cellular events cascading to a total cellular response. Interestingly, here we show that EVI1 is phosphorylated and the unphosphorylated form of EVI1 failed to regulate ΔNp63 that eventually showed no change in cell cycle/cell proliferation pattern. Our findings for the first time identify a possible link between two diverse signal transduction pathways acting together in response to a post-translationally modified oncogene that eventually delays cell cycle progression and inhibits cell proliferation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1301. doi:1538-7445.AM2012-1301
Abstract 1302: Effect of intestinal-specific Kaiso overexpression on the APC MIN/+ mouse colon cancer model
Authors: Wojtal Daria , Chaudhary Roopali , Nanan Kyster K. , Morone Simona , Daniel Juliet M.
Published: August 2012

The canonical Wnt signaling pathway is a crucial regulator of various cellular processes during embryogenesis but when aberrantly activated, it leads to tumorigenesis. The key downstream effector of the Wnt pathway is the Armadillo protein, β-catenin, which was first characterized as a cytoplasmic cofactor for the transmembrane, cell-cell adhesion tumor suppressor, E-cadherin. Recent evidence from our lab and others indicates that another E-cadherin cofactor, p120ctn and its binding partner, the POZ-ZF transcription factor Kaiso, co-regulate a subset of Wnt/β-catenin target genes and antagonize Wnt signaling. Specifically, using a Xenopus model system, we and others found that Kaiso overexpression rescued the β-catenin-induced duplicate axis phenotype; this implicated Kaiso as a negative regulator of Wnt signaling. However, it remains to be determined whether an analogous antagonistic role for Kaiso is conserved in mammals. Indeed, when Kaiso-null mice (which were viable and had no overt developmental defects or tumors) were crossed with the APCMIN/+ mouse model of colon cancer, the progeny had delayed tumor onset and reduced polyp size. This suggested that Kaiso played a positive role in tumor initiation and possibly cell proliferation in mammals. To resolve this discrepancy, we generated intestine-specific Kaiso overexpressing transgenic mice (KaisoTg/+) for subsequent mating with the APCMIN/+ mouse model of colon cancer. Histological analysis of small and large intestines from KaisoTg/+ and non-Tg mice revealed no gross morphological differences. Immunohistochemical (IHC) and immunofluorescence (IF) staining revealed strong nuclear Kaiso staining in intestinal tissues from KaisoTg/+ mice whereas intestinal tissues from non-Tg mice displayed weak cytoplasmic or no Kaiso staining. We detected no difference in β-Catenin expression levels in tissues from KaisoTg/+ mice and their wildtype littermates. This suggests that Kaiso's regulation of WNT signaling is downstream of its main effector β-Catenin. KaisoTg-APCMIN/+ double mutant mice have been generated and surprisingly they display decreased body size and weight, and have a decreased life span when compared to the parentals (i.e. KaisoTg or APCMIN/+). Current studies are aimed at assessing polyp formation and elucidating the expression levels of p120ctn, β-catenin, E-cadherin and cyclinD1 in the KaisoTg-APCMIN/+ intestines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1302. doi:1538-7445.AM2012-1302
Abstract 1303: The Wilms’ tumor gene, WT1, regulates E-cadherin expression and migration of prostate cancer cells.
Authors: Brett Adina , Fraizer Gail
Published: August 2012

Carcinoma of the prostate is one of the most common malignancies and second leading cause of death among American men. Even though patients with localized disease have high survival rates, patients with detectable metastasis have a median survival of only 12-15 months. One key step in the metastatic process is the loss of E-cadherin expression associated with tumor invasion. However the regulation of E-cadherin gene expression in prostate cancer (PrCa) cells is not well understood. Recently, the zinc finger transcription factor, WT1 was shown to regulate E-cadherin in NIH 3T3 cells and epicardial cells. We have previously reported the presence of WT1 protein in high grade prostate tumor sections, with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, the significance of WT1 expression in prostate cancer has not been established. The objective of this study was to determine whether WT1 might contribute to increase migration of PrCa cells by repressing E-cadherin expression. Using quantitative real-time PCR (QRTPCR), WT1 and E-cadherin mRNA levels were measured and found to be inversely related in PrCa cells. Similarly, QRTPCR results showed a decrease in E-cadherin mRNA in GFP/WT1 transfected PrCa cells, while E-cadherin levels were increased in PrCa cells with suppressed WT1 expression. Titration of WT1 levels also affected the migration of PrCa cells in wound healing and transwell migration assays. Silencing of WT1 by siWT1 RNA decreased the migratory potential of PC3 cells and the overexpression of GFP/WT1 increased the migration of LNCaP cells. A bioinformatics approach was used to identify potential WT1 binding sites in E-cadherin promoter, then direct in vivo binding of WT1 to the E-cadherin promoter in the chromatin of PrCa cells was confirmed by Chromatin Immunoprecipitation. Transcriptional control was quantified by co-transfection of a GFP/WT1 expression construct with E-cadherin promoter constructs. Results showed that WT1 decreased the activity of the proximal E-cadherin promoter in PC3 cells and DU145 cells. Using site directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for the repression of the E-cadherin proximal promoter by WT1. Overall these results demonstrate that WT1 transcriptionally down-regulates E-cadherin expression and thereby increases migration of PrCa cells. This finding suggests that the biological significance of WT1 expression in high grade prostate cancer lies in its contribution to cell migration and potentially to the promotion of metastasis. Supported by NIHR15CA11360 and the Ohio Board of Regents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1303. doi:1538-7445.AM2012-1303
Abstract 1427: Signal rewiring induced by EWS/FLI-1 in mouse and human mesenchymal stem cells
Authors: Marques Michelle , Nieves Bethsaida , Chen Ron , Vaka Dideepya , Chan Charles , Zheng Grace , Chang Howard , Weissman Irv , Sweet-Cordero Alejandro
Published: August 2012

Translocations involving members of the Ets family of transcription factors are common in prostate cancer and sarcomas, yet their effects on signal rewiring in cancer are unclear. Translocations between EWS and the Ets transcription factor Fli1 are found in Ewing's sarcoma, an aggressive tumor found in children and young adults. To elucidate the mechanism of transformation by EWS-Fli1, we have developed a novel approach that leverages the advantages of both mouse and human model systems. A knock-in mouse in which a Fli1-cDNA was introduced downstream of the endogenous EWS allele was generated. To our knowledge, this is the first described genetically engineered model in which EWS-Fli1 (EF) can be expressed conditionally from the native EWS promotor. Expression of EF in MEFs and MSCs from this model demonstrates that MSCs tolerate EF expression better than MEFs, consistent with prior data suggesting that MSCs are the likely cell of origin of Ewing's sarcoma. Ongoing analysis of well-defined MSC subsets may provide additional information regarding the cell of origin. In parallel, we have analyzed the consequences of expression of EF in human MSCs derived from pediatric patients. Deep-sequencing of these human pediatric MSCs (hpMSCs) has identified many novel coding and non-coding transcripts that are specifically upregulated in hpMSCs as a consequence of expression of EF. We have used functional analysis using hMSC and ES cell lines to validate the role of these genes in EWS pathogenesis. Some, but not all of these novel transcripts are also upregulated in mouse MSCs and MEFs that express EF. In summary, a cross-species approach that combines mouse genetics with functional genomics has identified novel targets for ES specific therapeutic development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1427. doi:1538-7445.AM2012-1427
Abstract 1430: Characterization of the medulloblastoma splice-ome reveals subgroup-specific changes in alternative splicing and isoform expression patterns
Authors: Dubuc Adrian M. , Morrissy Sorana , Kloosterhof Nanne , Northcott Paul , Yu Emily , Peacock John , French Pim , Taylor Michael
Published: August 2012

Medulloblastoma candidate gene approaches have previously identified alternative splicing and isoform expression changes affecting genes critical for cerebellar development and medulloblastoma pathogenesis. Here we present a bioinformatic characterization of the medulloblastoma splice-ome in 103 primary tumors and 14 normal cerebella samples. Medulloblastomas display a statistically significant increase in alternative splicing relative to normal fetal cerebella (2.3-times; P<6.47E-8), with splicing patterns that are specific to each molecular subgroup. Unsupervised hierarchical clustering of alternatively spliced genes accurately assigns medulloblastomas to their correct subgroup. One-third of all medulloblastomas (n=26) display a ‘hyper-spliced’ phenotype, with median splicing levels four-times greater than non-hyperspliced tumors. Hyperspliced medulloblastomas show a relatively worse overall survival (P<3.08E-2), and are seen across all molecular subgroups. Our analysis identifies previously reported splicing events targeting ERBB4 (mixed MB), GLI1 (SHH) and PTCH1 (SHH), supporting our approach. Subgroup-specific pathway analysis of alternatively spliced genes reveals extensive deregulation of neuronal pathways in non-WNT medulloblastomas, with the specific targeting of genes important for axonal guidance, synaptic transmission and neuronal differentiation. Finally, we present evidence for putative regulation of alternative splicing by antisense transcription. These data further demonstrate the differences between medulloblastoma subgroups, and highlight alternative splicing and isoform expression as a mechanism contributing to the transcriptional heterogeneity between subgroups, and perhaps to subgroup specific pathogenic mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1430. doi:1538-7445.AM2012-1430
Abstract 1432: The heterogeneous genomic landscape of posterior fossa ependymoma
Authors: Witt Hendrik , Mack Stephen C. , Bender Sebastian , Ryzhova Marina , Martin Sill , Isserlin Ruth , Jones David T. W. , Northcott Paul A. , Till Milde , Remke Marc , Benner Axel , Hielscher Thomas , Gupta Nalin , Garcia Livia , Weiss William A. , Betrand Kelsey , van Meter Timothy , von Deimling Andreas , Rutkowski Stefan , Pietsch Torsten , Kulozik Andreas E. , Cho Yoon-Jae , Tabori Uri , Hawkins Cynthia E. , Witt Olaf , Bader Gary D. , Rutka James T. , Lichter Peter , Korshunov Andrey , Taylor Michael D. , Pfister Stefan M.
Published: August 2012

Brain tumors are the most common cause of cancer-related death in childhood. Ependymomas, are the third most common pediatric brain tumor. The disease remains incurable for about 45% of patients even after gross total resection and radiotherapy. Despite showing a very homogeneous histological picture, ependymomas display distinct molecular behavior, which supports the existence of several independent entities of the disease. We examined two non-overlapping cohorts of 102 and 75 ependymomas by mRNA expression profiling, on two different array platforms (Affymetrix, Agilent). When performing multiple statistical clustering methods (unsupervised consensus NMF and consensus HCL), we could consistently identify three major clusters, including two subgroups of posterior fossa (PF) ependymoma, a variant common in children and associated with heterogeneous clinical outcome. Subgroup-specific chromosome aberrations of PF tumors were detected by aCGH, and biological signaling pathways distinguishing PF subgroups were identified by gene set enrichment analysis and visualized in Cytoscape. We validated the most significantly classifying markers of each subgroup by immunohistochemistry on a tissue microarray containing an independent set of 265 PF ependymomas. Our findings delineate two subgroups of PF ependymoma (groups A and B) which are demographically, transcriptionally, genetically, and clinically distinct. Group A patients are younger, have laterally located tumors with a balanced genome, more frequently develop secondary metastases and are much more likely to have an extremely poor outcome as compared with group B patients. Based on a multi-variate Cox proportional-hazards model, our identified markers have the strongest independent prognostic value among demographic and molecular variables with Hazard ratios of 8.45 (PFS) and 10.55 (OS). Prognostic significance and predictive impact is being validated in the GPOH HIT2000 Ependymoma study. The identification of two distinct subgroups of PF ependymoma, and markers applicable for their clinical distinction, will allow for better prognostication of individual cases, independent of age, level of resection and WHO grade, and also for stratification in future ependymoma clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1432. doi:1538-7445.AM2012-1432
Abstract 1448: Whole genome RNAi screen identifies proteasome inhibition as a strategy for NSCLC radiosensitization
Authors: Zhu Kaya , Kushwaha Deepa , Cron Kyle , Hsieh Grace , Merzon Dmitry , Evans Daniel , Ng Kimberly , Chen Clark C. , D'Andrea Alan D. , Kozono David
Published: August 2012

Background: Each year, approximately 60,000 patients in the US are diagnosed with locally advanced non-small cell lung carcinoma (NSCLC). The majority is treated with a combination of radiation therapy (RT), chemotherapy +/− surgery. Despite best available therapy, over a third of patients fail locoregionally. The addition of concurrent chemotherapy to RT adds only about a 5% absolute survival benefit. Novel radiosensitizers may improve the therapeutic index of RT. Materials and Methods: We performed whole genome pooled RNAi screens using the Hannon-Elledge library of 74,705 distinct shRNAs directed against over 18,000 genes, in two NSCLC cell lines A549 and NCI-H460. After stable transduction, we passaged the lines for 12 population doublings, and extracted DNA both before and after passaging to compare the representation of each shRNA at each time point. We defined top hits as those genes for which multiple shRNAs decreased in representation by at least 50% during passaging, signifying silencings that were detrimental to proliferation. Results: The top hit of our screen was PSMA1, a subunit of the 20S proteasome; out of six shRNAs, between four and six scored in the two lines in screens performed in triplicate. Bortezomib, an inhibitor of the 20S proteasome, has shown activity against NSCLC in preclinical studies and Phase I/II clinical trials. We observed that bortezomib results in synergistic cytotoxicity in cells treated with fractionated ionizing radiation (IR) in vitro. 10 nM bortezomib resulted in 46% clonogenic survival of unirradiated A549 cells, but only 29% survival of cells treated with 1 Gy x 3 daily fractions compared to cells treated with IR alone. Similar results were observed with MG-132 and with NCI-H460. In soft agar anchorage-independent growth assays, bortezomib resulted in less than 1% colony formation following 1 Gy x 3 daily fractions compared to cells treated with IR alone. In vivo experiments are underway with subcutaneous xenografts in nude mice treated with concurrent CT-guided conformal RT. A retrospective analysis of 443 patients with profiled lung adenocarcinomas showed worse overall survival among patients with tumors showing high proteasome gene expression (median OS 4.2 vs. 6.2 y, 5-year OS 47.5 vs. 61.0%, log rank p = 0.04). Conclusions: Proteasome inhibition emerged as the top drug target of an RNAi screen designed to identify targets with sustained activity over multiple cell generations. Its synergistic impact on NSCLC viability when coupled with fractionated RT should be further explored in preclinical studies and early clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1448. doi:1538-7445.AM2012-1448
Abstract 145: MicroRNA-23b Expression is Regulated by VHL and effects on Glioma Cell survival and invasion
Authors: Han Lei , Chen Lingchao , Zhang Kailiang , Shi Zhendong , Zhang Junxia , Zhang Anling , Wang Yongzhi , Song Yijun , Zheng Yongri , Jiang Tao , Pu Peiyu , Jiang Chuanlu , Kang Chunsheng
Published: August 2012

Background Glioblastoma is the most common malignant primary brain tumor in adults. While increasing evidence indicates that the deregulation of miRNAs is involved in a wide range of human cancers, the roles and potential mechanisms of miRNA in glioma development and progression are still largely unknown. Methods The expression of miR-23b in 48 clinical samples and five glioma cell lines with different grades was examined by FISH and qRT-PCR. Flow cytometry, Annexin V method, Colony formation assay, and Transwell cell invasion test were used to detect the proliferation and invasive activity of the tumor cells before and after transfection with AS-miR-23b of glioma cells. Luciferase assay and western blot analysis were used to examine that VHL is a direct target of miR-23b. In vivo anti-glioma effect of AS-miR-23b was investigated using subcutaneous xenograft models. Results We identified that expression of miR-23b is elevated in both glioma tissue samples and glioma cells by real-time PCR and FISH analyses. Knock-down of miR-23b expression triggered growth inhibition, induced apoptosis and suppressed invasion of glioma cells in vitro. Luciferase assay and western blot analysis revealed that VHL is a direct target of miR-23b. As such, restored expression of VHL inhibited glioma cell survival and induced apoptosis. Mechanistic investigation revealed that miR-23b deletion decreased HIF-1α/VEGF expression and suppressed β-catenin/Tcf-4 transcription activity in a VHL-dependent manner. Furthermore, expression of VHL was inversely correlated with miR-23b in glioblastoma samples and was predictive of patient survival in a retrospective analysis. Conclusion In summary, we demonstrate that down-regulation of miR-23b inhibits survival and invasion of glioma by repressing expression of VHL. Genetic restoration of VHL bypassed the effects of miR-23b overexpression, suppressing the ranscriptional activities of both the HIF/VEGF and β-catenin/Tcf-4 pathways. Based upon these observations, we propose that anti-angiogenic therapy for glioma could benefit from adjuvent miRNA therapy targeting the HIF1α and β-catenin/Tcf-4 pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 145. doi:1538-7445.AM2012-145
Abstract 1451: DAB2IP, a novel tumor suppressor protein modulates prostate cancer cell death in response to the combined treatment of ionizing radiation and a DNA-PKcs inhibitor NU7441
Authors: Yu Lan , Hsu Feng-Ming , Hsieh Jer-Tsong , Saha Debabrata
Published: August 2012

Introduction: Prostate cancer (PCa) is the most common malignancy (25%) and the second leading cause of cancer-related death (10%) in U.S. men. Radiation treatment, which is noninvasive and well tolerated, is one of the front line therapeutic interventions in the management of localized PCa. However, significant proportions of high-risk patients fail therapy and develop metastatic disease. DAB2IP, which is a novel member of the Ras GTPase-activating protein family and a regulator of PI3K/Akt activity, is often downregulated and associated with aggressive prostate cancer (PCa). The purpose of this study is to investigate the radio-sensitizing property of a novel DNA-PKcs inhibitor, NU7441, in DAB2IP- deficient human PCa cells. Methods: We have developed DAB2IP deficient and proficient PCa cell lines (C4-2, PC3) for this study. We performed clonogenic surviving fraction analysis, DNA double strand break repair kinetics, cell cycle analysis, TUNEL assays, immunofluorescence, immunoblot, apoptosis and autophagy assays to investigate the mechanism of radiosensitization of NU7441 in these PCa cells. Results: Clonogenic surviving fraction analysis indicates a strong radiosensitizing ability of NU7441 in PCa cells deficient in DAB2IP. This is primarily due to robust G2-M block and persistent DNA double strand break in response to the combined treatment of radiation and NU7441. We clearly demonstrate that the presence of DAB2IP modulates prostate cancer cell death when treated with radiation and NU7441. Apoptotic cell death was prominent in PCa cells proficient in DAB2IP whereas, robust autophagic cell death was noticed in DAB2IP deficient PCa cells. Conclusion: Our study clearly shows that NU7441, a novel DNA-PKcs inhibitor, is a potent radiosensitizer in aggressive PCa cells. Furthermore, our study indicates that DAB2IP may act as an important factor in PCa cell death after combined treatment Nu7441 and radiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1451. doi:1538-7445.AM2012-1451
Abstract 1456: Sulfasalazine sensitizes glioblastoma cells to radiation treatment
Authors: Sleire Linda , Wang Jian , Heggdal Jan , Pedersen Paal-Henning , Enger Per Øyvind
Published: August 2012

Glioblastoma (GBM) is a lethal cancer with a limited response to ionizing radiation. Recent studies suggest that Sulfasalazine (SAS), a drug used to treat inflammatory bowel disease, inhibits the Xc- antiporter system in glioma cells, thereby blocking their uptake of cystein. Since the availability of cystein is a rate limiting step in intracellular antioxidant production, we wanted to investigate whether sulfasalazine sensitizes glioma cells to radiation. Expression of xCT, the catalytic subunit of system Xc-, was found in 30 patient GBM biopsies. SAS effect on glioma cell growth was investigated using an electric cell substrate impedance sensing (ECIS) instrument. All glioma cell lines showed altered growth curves in response to SAS treatment. To assess the effect of blocking the antiporter, intracellular levels of the antioxidant glutathione were measured. With increasing doses of SAS, glutathione levels decreased in a dose response manner. In addition, cysteine was added to the medium to see if the cells could survive high doses of SAS. U251 glioma cells were treated with escalating doses of SAS, alone or in combination with radiation (8 Gy). Nuclear integrity was evaluated to estimate cell death following treatment, as well as the presence of double stranded breaks. In addition, cell death and viability were investigated using live/dead staining and the MTS assay. All treatment groups exhibited increased rates of cell death compared to untreated controls. A combination of SAS and radiation resulted in higher levels of cell death, than radiation or SAS administered alone. In order to assess whether this can be exploited therapeutically, we are preparing to treat nude rats harbouring glioblastoma biopsy xenografts with SAS, alone or in combination with gamma knife radiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1456. doi:1538-7445.AM2012-1456
Abstract 146: MicroRNA-150 expression induces differentiation of myeloid leukemias
Authors: Morris Valerie A. , Zhang Ailin , Oehler Vivian G.
Published: August 2012

Blocked myeloid differentiation is a major feature of chronic myeloid leukemia (CML) disease progression and acute myeloid leukemia (AML). Normal myeloid differentiation is a tightly controlled process involving the coordinated action of specific transcription factors, which are dysregulated by chromosomal translocations, DNA mutations, epigenetic changes, and aberrant gene and microRNA (miRNA) expression in leukemias. Previously, we identified decreased miR-150 expression in CML and AML cell lines and primary cells when compared to normal bone marrow, and determined miR-150 expression is decreased in CML by the Bcr-Abl fusion protein. Furthermore, lentiviral expression of miR-150 in AML cell lines and primary CML patient samples induced myeloid differentiation as detected by CD11b expression, morphologic differentiation, and colony forming ability in methylcellulose, both in the absence and presence of the differentiating agents. In this study, we elucidated the targets responsible for miR-150 induced differentiation. Expression of the validated miR-150 target MYB, a transcription factor involved in blocking differentiation in progenitor cells, was only decreased in miR-150 expressing cells in the presence of differentiating agent all-trans retinoic acid (ATRA). Furthermore, lentiviral overexpression of MYB lacking miR-150 binding sites in the 3′-UTR partially decreased the CD11b expression in the presence and absence of ATRA in both control and miR-150 expressing HL60 cells. These results suggest additional miR-150 targets are involved in differentiation besides MYB. To identify additional miRNA-150 targets, we profiled changes in mRNAs associated with the RNA-induced silencing complex (RISC) using RNA-immunoprecipitation of Ago2 followed by microarray gene expression profiling in miR-150 versus control HL60 cells. Furthermore, although almost all functional investigations have focused on the mature strand, we have found that miR-150 and its partner star strand miR-150* are expressed at almost equal levels in normal myeloid hematopoietic cells, and are investigating the individual contributions of their separate targets in induction of differentiation. In conclusion, miR-150 expression promotes myeloid differentiation through targeting transcription factor MYB and additional targets, while downregulation of miR-150 in myeloid leukemias contributes to the block in differentiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 146. doi:1538-7445.AM2012-146