bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-211: Identification of blood-based protein biomarker candidates for malignant glioma
Authors: Xu Baogang J. , An Qi A. , Gowda Supriya , Yan Wenwei , Abel Ty , Pierce Larry , Weaver Kyle D. , Thompson Reid C.
Published: February 2011

A reliable, non-invasive and convenient method for detecting malignant gliomas progression and responsiveness to treatment will significantly increase the survival of patients. A protein biomarker-based method for high-grade glioma using patients’ blood samples was investigated. Using a multiplex Luminex immunoassay, 90 proteins associated with cancer were examined in the plasma from high-grade glioma patients (N=28) and non-cancer controls (N=367). Wilcoxon rank-sum test was performed to select the differentially expressed proteins. Receiver operating characteristics (ROC) curves were constructed to determine their diagnostic accuracy. A second cohort of serum samples from different GBM patients (N=20) and non-cancer controls (N=1278) was similarly analyzed. A total of 40 and 25 proteins were found to be significantly differentially expressed between the glioma and control groups (p < 0.0005) using the plasma or serum samples, respectively. With ROC analyses, 19 and 9 proteins were found with area under the curve greater than 85% using the plasma or serum samples, respectively. Five protein biomarkers with an average of 87.2% sensitivity and 87.4% specificity were similarly identified in these two different cohorts of patients. In addition, four plasma protein biomarkers were found to be significantly associated with high-grade glioma patients’ survival. Immunohistochemistry (IHC) showed that human glioblastoma tissue positively expressed the identified protein biomarkers. Differential expression levels for one of the identified protein biomarkers, MIP 1-alpha, were further investigated using human glioma tissue microarray. Compared to grade II glioma tissues (N=22), MIP 1-alpha was found to be overexpressed in grade III (N=9) and grade IV (N=41) glioma tissues (p < 0.0001). Furthermore, MIP 1-alpha was found to directly promote T98G glioma cell line proliferation and migration (p<0.05) using WST-1 proliferation assay and wound closure assay, respectively. Overall, a blood-based protein signature for high-grade gliomas was identified which may offer a minimally-invasive approach for diagnosing high-grade glioma and monitoring for recurrence or response to therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-211.
Abstract LB-215: Hypoxia inducible factor-1 alpha (HIF-1α) splice variants: Potential prognostic biomarkers in breast cancer
Authors: Gabert Jean A. , Beaufils Nathalie , Formisano-Tréziny Christine , Picard Christophe , Silvy Monique , Giusiano Sophie , Charpin Colette , Pauly Vanessa , Pradel Vincent , Bonnier Pascal , Dales Jean-Philippe
Published: February 2011

Background: Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. HIF-1 protein is composed of two HIF-1α and HIF-1ß/ARNT subunits. Prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variants expression on breast cancer prognosis in term of metastasis risk is not well known.

Methods: With real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and four variants in breast specimens of a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumor microvessel density and metastasis-free survival.

Results: HIF-1α isoforms containing three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1 TAG) and HIF-1 736mRNAs were found expressed at higher levels in estrogen receptor negative carcinomas compared to normal/benign tissues (p=0.009 and p=0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1 TAG mRNA levels (p=0.037). Significant statistical association was found between tumor grade and HIF-1 TAG (p=0.048), and total HIF-1 (p=0.048) mRNA levels. HIF-1 TAGmRNA levels also correlated inversely with both estrogen and progesterone receptor status (p=0.005 and p=0.033 respectively). Univariate analysis showed that high HIF-1 TAG mRNA levels correlated with shortened metastasis free survival (p=0.01).

Conclusion: Our results show for the first time that mRNA expression of HIF-1 TAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-215.
Abstract LB-22: Repression of alpha-, beta- and gamma-catenin genes by SNAI2 in human breast cancer cells
Authors: Mittal Mukul K. , Misra Smita , Bailey Charvann K. , Chaudhuri Gautam
Published: February 2011

SNAI2 is a transcriptional repressor protein implicated in the proliferation and metastasis of several human cancer cells. It binds to the E2-box sequence of its target gene promoters and down regulates their expressions by chromatin remodeling. While studying SNAI2-binding gene promoters in the human breast cells by ChIP-DSL technique, we found that SNAI2 binds to the promoters of the cytoskeleton proteins alpha-, beta- and gamma-catenins. These proteins help cells to maintain their epithelial phenotype and apico-basal polarity. Reduced expression of these proteins has been shown by IHC in high grade metastatic breast carcinoma. Here we report the molecular characterization of this repression. Highly invasive breast cancer cells (MDA-MB-231 and BT-549) express high levels of SNAI2 while mildly invasive (MCF 7 and MDA-MB-468) cells do not express SNAI2. Over-expression of SNAI2 in the SNAI2-negative MCF7 and MDA-MB-468 cells down regulated the mRNA and protein levels of the catenins in these cells. siRNA-mediated knock down of SNAI2 in the SNAI2-positive MDA-MB-231 and BT549 cells elevated the mRNA and protein levels of these proteins in these cells. SNAI2 over-expression induces the invasion and migration in non invasive cells, while down-regulation of the SNAI2 decreases the invasion and migration potential of highly invasive breast cancer cells. Chromatin immunoprecipitation data revealed co-recruitment of CtBP1 and HDAC1 at the catenin gene promoters in the SNAI2-expressing cells further indicating that SNAI2 represses these genes through chromatin remodeling. Our study thus adds to the mode of action of SNAI2 in mediating metastatic conversion of human breast cancer cells. Supported by the DOD-CDMRP IDEA Grant# W81XWH-06-1-0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-22.
Abstract LB-233: IGF activation in a molecular subclass of hepatocellular carcinoma and preclinical efficacy of IGF-1R blockage
Authors: Tovar Victoria , Alsinet Clara , Villanueva Augusto , Hoshida Yujin , Chiang Derek Y. , Solé Manel , Thung Swan , Moyano Susana , Toffanin Sara , Minguez Beatriz , Cabellos Laia , Peix Judit , Schwartz Myron , Mazzaferro Vincenzo , Bruix Jordi , Llovet Josep M.
Published: February 2011

Background: IGF signaling contributes to proliferation and cell survival through PI3K/AKT and MAPK pathways. It has a relevant role in a variety of human malignancies.

Aims: (1) To analyze the role of IGF signaling in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and (2) To asses the antitumoral activity of IGF pathway targeted therapies.

Methods: An integrative molecular dissection of the axis was performed in a cohort of 104 HCCs, including gene (quantitative PCR and Affymetrix U133 2.0® microarray) and miRNA expression (ligation-mediated amplification method), structural aberrations (Affymetrix STY Mapping Array®) and protein activation (immunohistochemistry). The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro through viability and proliferation assays. A xenograft model of HCC was used to evaluate A12 activity in vivo.

Results: Activation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 -resulting from reactivation of fetal promoters-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with IGF2 overexpression (p=0.03), mTOR signaling (p=0.035) and was enriched in a molecularly distinct HCC subclass defined through unsupervised hierarchical clustering of the same cohort of samples (Proliferation subclass) (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR<0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 treated mice exhibited delayed tumor growth and prolonged survival in comparison to control mice, a reduction in proliferation rates and induction of apoptosis was observed in treated mice tumoral samples.

Conclusions: Integrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-233.
Abstract LB-242: Oncosomes: Large bioactive particles secreted by prostate cancer cells
Authors: Vizio Dolores Di , Morello Matteo , McNeish Brendan , Schow Peter , Lisanti Michael P. , Moses Marsha A. , Freeman Michael R.
Published: February 2011

Purpose of the study: Human prostate tumor cells were recently shown by our group to secrete large (0.5-10 m diameter) bioactive microvesicles capable of activating signal transduction and inducing proliferation and migration in cells in the tumor microenvironment (Di Vizio, et al. Cancer Res. 2009 69: 5601-9). These secreted particles, termed oncosomes, were shown to be vehicles for numerous signaling proteins involved in cell growth and motility. They also contain the membrane protein caveolin-1 (Cav-1), a mediator of prostate cancer (PCa) metastasis and a circulating PCa biomarker. The purpose of this study was to develop the tools to identify, isolate, sort and characterize PCa-derived oncosomes.

Experimental procedures: Secreted particles from PCa cells were ultracentrifuged at 100,000xg and the pelleted material processed on a Moflo High-Speed Cell Sorter (Beckman-Coulter) and analyzed using Summit V4.3 software (Dako Colorado, Inc.). 1, 3 and 10 m bead standards (Spherotech Inc.) were used to set size gates. To avoid capturing aggregated material, forward scatter and pulse width were set at a linear scale and only single events were gated and analyzed. Sorted particles were visualized by confocal and electron microscopy.

Results: Using the above procedure, we were able to retrieve intact particles of 1-10 m in diameter secreted by LNCaP cells overexpressing the oncoprotein MyrAkt1. The nontumorigenic prostate stromal cell line WPMY-1 secreted > 10-fold fewer particles in this size range. We confirmed that these were membrane-derived particles by showing that they contained MyrAkt1, which localizes to the plasma membrane. Particles in a similar size range were also sorted after purification from LNCaP cells stably expressing a Cav-1/GFP fusion protein. Sorted particles visualized microscopically appeared to be surrounded by a lipid bilayer and to have intrinsic structure that made them resistant to collapse. Particles isolated by these methods evoked degradation of FITC-labeled collagen and contained active MMP9 and MMP2, two key proteases involved in tumor cell invasion.

Conclusions: The specific type of large bioactive particle we have isolated has not been previously described in any tumor system. Studies are ongoing to detect these particles in animal models of cancer and in human patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-242.
Abstract LB-267: GRB7: A novel target in basal breast cancer
Authors: Giricz Orsi , Pero Stephanie C. , Krag David N. , Sparano Joseph A. , Kenny Paraic A.
Published: February 2011

Basal breast cancers lack expression of estrogen and progesterone receptors and HER2, rendering them non-responsive to the most commonly used targeted therapies in breast cancer. These tumors are typically more aggressive, more locally invasive and more likely to metastasize to the brain than other breast cancer subtypes and, consequently, have a poor prognosis. GRB7 is a cytoplasmic adaptor protein that interacts with receptor tyrosine kinases, relaying information to their downstream signaling cascades. It also plays a role in integrin signaling and cell migration by binding focal adhesion kinase (FAK) and ephrin receptors. It is a part of the HER2 amplicon and is frequently overexpressed in HER2-amplified breast tumors. Breast tumors in which HER2 and GRB7 are both co-amplified and co-overexpressed have a poor prognosis.

We and others have recently determined that high levels of GRB7 are associated with recurrence in women with triple-negative breast cancer treated with anthracyclines (Sparano et al. J Clin Oncol 27(15s) 500). In this study, we have tested the hypothesis that triple-negative breast cancer cell lines are dependent on GRB7 for proliferation, migration and invasion. We have utilized a highly specific cell-penetrating peptide inhibitor of GRB7 (G7-18NATE) and tested its effects on a panel of 5 basal breast cancer cell lines in a series of culture assays: monolayer wound healing assays (motility on 2D substrates), transwell invasion assays (in vitro invasiveness) and 3D Matrigel cultures (morphogenesis in 3D environment). In all cases evaluated, the Grb7 inhibitory peptide impaired the proliferation, migration and invasion of these cell lines, even though these cells express low levels of GRB7 compared to HER2-amplified cell lines.

These data implicate GRB7 as a key mediator of several important phenotypes of basal breast cancer cells and suggest that GRB7 itself, or GRB7-dependent pathways, may prove to be important targets for the development of specific therapies for this disease.

This work was supported in part by Susan G. Komen for the Cure (KG091136).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-267.
Abstract LB-268: RTVP-1 regulates glioma cell migration and podosome formation via interaction with N-WASP
Authors: Lee Jae Kyung , Av Amotz Ziv , Xiang Cunli , Cazacu Simona , Finniss Susan , Giladi Nis , Barda-Saad Mira , Brodie Chaya
Published: February 2011

Gliomas, the most frequent primary brain tumor are characterized by increased proliferation and invasion into the surrounding normal brain tissue. In a recent study we reported that RTVP-1 is highly expressed in gliomas and that its expression is correlated with the degree of malignancy of astrocytic tumors. In addition, RTVP-1 is involved in the regulation of the growth, survival, and migration of glioma cells. To further delineate the molecular mechanisms underlying the effect of RTVP-1 in glioma cells we performed a pull down assay using His-tagged RTVP-1 followed by mass spectroscopy, and found that RTVP-1 was associated with the actin polymerization regulator, N-WASP. The association of RTVP-1 and N-WASP was further validated by co-immunoprecipitation and FRET analysis. Since N-WASP regulates cell spreading and migration we examined the role of RTVP-1 in these processes. We found that overexpression of RTVP-1 increased, whereas silencing of this protein decreased cell spreading and migration of glioma cells. The effect of RTVP-1 was mediated by N-WASP since silencing of N-WASP abolished RTVP-1 effects. In addition, RTVP-1 increased podosome formation in glioma cells and this effect was also mediated via its interaction with N-WASP. Moreover, the association of RTVP-1 and N-WASP was increased during cell spreading and appeared to be localized to contact areas of the spreading cells with the extracellular matrix. Another protein which was found by the pull down assay to interact with RTVP-1 is hnRNPK. This protein has been recently reported to associate with and to inhibit the effect of N-WASP on cell spreading. Using co-immunoprecipitation and FRET analysis we validated the interactions of hnRNPK with N-WASP and RTVP-1 in glioma cells. In addition, using co-immunoprecipitation studies, we found that overexpression of RTVP-1 decreased the association of N-WASP and hnRNPK, therefore suggesting that RTVP-1 may activate N-WASP by decreasing its association with hnRNPK. In summary, we report that RTVP-1 regulates glioma cell spreading, migration and invasion and that these effects are mediated via interaction with N-WASP and by interfering with the inhibitory effect of hnRNPK on the function of N-WASP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-268.
Abstract LB-276: A novel approach using the telomerase-selective adenoviral marker (OBP-401) for detection of circulation tumor cells in breast cancer patients
Authors: Kim Seung Jin , Sato Jun , Taguchi Tetsuya , Tamaki Yasuhiro , Tsukamoto Fumine , Akazawa Kenji , Masago Akinori , Tsujino Yukio , Urata Yasuo , Noguchi Shinzaburo
Published: February 2011

Background: The detection of circulating tumor cells (CTCs) is a potential method to predict survival of metastatic breast cancer patients as well as outcomes of early breast cancer patients. However, no method for CTCs has yet proven to be the golden standard. We developed a new approach for detecting CTCs using the telomerase-selective adenoviral marker (OBP-401, Oncolys Biopharma). This marker contains the replication cassette, in which the human telomerase reverse transcriptase promoter drives expression of E1 genes, and the green fluorescent protein (GFP) gene for monitoring viral replication. Thus, OBP-401 infects viable cancer cells and replicates in them under the telomerase activity, and we can detect viable CTCs as GFP-positive cells.

Methods: This system is consisted of the following steps; (1) virus-infection for 7.5 ml whole blood (incubated with 4 × 10sup8 Plaque Forming Unit OBP-401 virus for 24 hours at 37 Celsius), (2) dead cell staining using L23102 (invitrogen), (3) virus-inactivation and RBC elimination, and (4) detection of GFP expressing cells using fluorescence microscopy. In a preclinical study, the sensitivity of this system was assessed using cell lines. Next, we conducted feasibility studies for CTCs detection in 80 healthy individuals, 70 metastatic, and 27 early breast cancer patients. In metastatic and early breast cancer patients, we compared the sensitivity of this system with that of CellSearch® (Veridex). GFP-positive cells (viable CTCs) and L23102 expressing cells measuring ≥ 20 m in diameter (dead CTCs) were considered as CTCs in this system.

Preliminary data: The sensitivity of this system, which was determined by a serial dilution of MDA-MB-468 cells against healthy volunteer's blood, was 1 cell per 7.5 ml. Neither viable nor dead CTCs were detected in any of healthy controls. Of 50 metastatic patients, 12% were primary breast cancers with stage IV disease, 24% were in the 1st line chemotherapy setting, and 42 % were heavily pretreated with chemotherapy. The sensitivities of tumor markers (CEA and CA15-3), CellSearch, and OBP-401 were 78%, 54%, and 66%, respectively.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-276.
Abstract LB-295: Identification of the first class of proteasome inhibitors that impair 26S proteasome assembly in vitro.
Authors: Etievant Chantal , Riquet William , Roullet Nicolas , Knibiehler Martine , Vandenberghe Isabelle , Carletti Isabelle , Gairin Jean Edouard
Published: February 2011

The Ubiquitin-Proteasome System (UPS) is the main non-lysosomial proteolytic machinery in the eukaryotic cells. Prior to their degradation proteins are adressed to the catalytic 26S proteasome complex composed of a 20S “core” that contains proteolytic activities, assembled with one or two 19S regulatory particles that bind proteins to be degraded. Due to its central role of in regulating cell cycle progression and signal transduction the UPS has been proposed as a suitable target for antineoplastic strategies. To date only catalytic inhibitors of proteasome, targeting proteolytic activities of the 20S core particle, have been characterized and/or have emerged as potent anticancer drugs (1).

However, the capacity of the UPS to degrade proteins not only depends of the catalytic activities of the 20S core, but also of the 26S proteasome assembly, a dynamic and tightly controlled cellular process. Thus, compounds impairing 26S proteasome assembly could represent a novel class of proteasome inhibitors.

With the aim of identifying such molecules we first set up a robust biochemical screening assay allowing us to follow 26S proteasome assembly in vitro. First, 26S proteasome was purified from Hela cells using affinity chromatography followed by gel filtration. Then, experimental conditions were determined for in vitro 26S disassembly into its constitutive sub-complexes (19S+20S) ands its re-assembly as an active and functional 26S entity. The differential CTL-activity between 26S and 20S proteasomes (2) was then used as the readout to follow the reassembly of 26S in vitro, prior to be confirmed using in gel overlay experiments and western-blotting. Using this biochemical assay:

1) we screened naturals compounds endowed with antiproliferative properties and described as inhibitors of the ubiquitine-proteasome pathway (3).

2) we identified structurally-related natural molecules, epidithiodioxopiperazine and diketodithiopiperazine derivatives, as the first class of molecules inhibiting proteasome assembly without significantly affecting neither 26S nor 20S catalytic activity in vitro.

Our research efforts now concentrate in showing that: 1) these molecules inhibit proteasome assembly in cells, and that 2) this inhibition can be correlated with their antiproliferative effects.

References

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-295.
Abstract LB-305: Why two are better than one: Suppression of carcinoma cell growth in vitro and in human HGF/SF transgenic SCID by inhibitors of MET (SGX523) and EGFR (erlotinib)
Authors: Zhang Yu-Wen , Staal Ben , Essenburg Curt , Su Yanli , Kang Liang , West Rich , Kaufman Dafna , DeKoning Tom , Eagleson Bryn , Buchanan Sean , Vande Woude George
Published: February 2011

The signaling cascade originating from hepatocyte growth factor/scatter factor (HGF/SF)-MET ligand-receptor pair is frequently observed in a broad spectrum of human cancers. Inappropriate activation of this pathway has been shown to occur via: paracrine/autocrine activation; by amplification or mutation in the MET gene; or through ligand independent mechanisms (www.vai.org/met). Various MET drugs targeting this pathway are under development and in need of relevant in vivo tumor models to allow assessment of drug efficacy alone or in combination against human MET expressing tumors. Conventional mouse models post a significant limitation on fulfilling such demand as mouse HGF/SF has low affinity/activity on the human MET. Such limitations are abrogated in human HGF/SF transgenic SCIDs (designated hHGFtg-SCID), which, unlike its non-transgenic littermates, produce human HGF/SF to allow human MET signaling on cells of human cancer xenografts. Here, we show that the hHGFtg-SCID mouse significantly enhances tumor xenograft growth of many MET-expressing human carcinoma cell lines including lung, breast, kidney, colon, gastric and pancreatic cancers. Using the hHGFtg-SCID mouse model systems, we demonstrated that SGX523, a MET small molecule kinase inhibitor which suppressed MET phosphorylation and downstream signaling activation in vitro, partially inhibits the growth of xenografts derived from NCI-H596 lung cancer cells, HCC1954 breast cancer cells and HPAF II pancreatic cancer cells. Furthermore, we found that the partial tumor growth inhibition by SGX523 was significantly enhanced by co-administration of erlotinib, the EGFR small molecule kinase inhibitor. In vitro, erlotinib also enhanced SGX523 inhibitory activity in cell proliferation and G1/S cell cycle progression assays. Our data suggest that the hHGFtg-SCID mouse is a valuable system for evaluating MET drugs, and simultaneously targeting MET and EGFR pathways may provide a better treatment for cancers harboring inappropriate activation of both pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-305.
Abstract LB-31: Drug combination studies: theory and analysis
Authors: Hixon Mark S.
Published: February 2011

A novel method for the graphical analysis of drug combination studies is presented. This method, Delta Plot Analysis, addresses the shortcomings of Isobolograms and Combination Index determinations. By use of this method, one may choose either a Bliss Independence or Loewe Additivity model and conduct the analysis. The method is comparative in nature, plotted as a function of cell survival (tracking other observables is also possible), and most importantly contains confidence intervals for determining significance above/below an additive effect line. In short, assay data with much scatter require greater differentiation from additivity to be interpreted as anything other than additive while data with less scatter allow one to conclude synergy or antagonism with less deviation from theoretical additive. Notably, the method handles partial response curves and makes use of single agent dose response curves to incorporate experimental uncertainty. A great advantage of the method is that synergistic and antagonistic interactions produce signature patterns in addition to providing data points falling outside of the additive effect confidence interval - something of great utility to the analyst. Also intriguingly, when accounting for experimental uncertainty (through simulations or with empirical data), a synergistic interaction is revealed to be synergistic regardless of the choice of Bliss or Loewe models. Delta Plot Analysis has been validated with published data and confirmed with in-house drug combination studies. The method is robust, requires no specialized software and easily depicts a multidrug combination study. This presentation also includes a look at the theory behind multiple drug interactions from a first principles perspective and provides a useful way of connecting drug action with drug effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-31.
Abstract LB-313: Soluble UL16 binding protein 2 is elevated in the sera of lung cancer patients
Authors: Yamaguchi Kosuke , Chikumi Hiroki , Kinoshita Naoki , Shimizu Asuka , Hashimoto Kiyoshi , Kurai Jun , Yamasaki Akira , Nakamoto Masaki , Matsumoto Shingo , Takata Miyako , Igishi Tadashi , Burioka Naoto , Shimizu Eiji
Published: February 2011

NKG2D ligands are the surface molecules which induce expression on transformed cells. Specifically they bind to NKG2D on NK cells, and this binding induces activation of NK cells. In humans, NKG2D ligands are described as MICA, MICB and ULBP1, 2, 3, and 4. In this study, we explored the clinical and immunological significance of NKG2D ligands on lung cancer. We evaluated the expression of each NKG2D ligand on the cell surface of various lung cancer cell lines by flow cytometory. The soluble form of ULBP2 was measured in culture supernatant and in sera of lung cancer patients by an original developed ELISA system. Immunological effects of cell surface ULBP2 and soluble form of ULBP2 were examined by 3.5-h 51Cr release assay.

We found that ULBP2 was the most widely and strongly expressed NKG2D ligand in lung cancer cell lines. Soluble form of ULBP2 was increased in culture supernatant of the lung cancer cell lines with high expression of cell surface ULBP2. In addition, soluble form of ULBP2 was detectable in the sera of lung cancer patients and increased amount of soluble ULBP2 in sera significantly correlated with poor prognosis lung cancer patients. We evaluated the the immunological effect of soluble ULBP2 separately using human peripheral blood mononuclear cells (PBMCs), and found that soluble ULBP2 suppressed NK activity.

In conclusion, ULBP2 is the most significant NKG2D ligand in lung cancer and its soluble form was detectable and elevated in sera of lung cancer patients. Because soluble form of ULBP2 is one of the determinant factor of the anti-tumor immunity of the cancer patients, the development of intervention in this effect would be a candidate for the promising immuno-modulatory therapy

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-313.
Abstract LB-320: Inhibition of TGFß1 improves the therapeutic efficacy of non-immunologic tumor cell based vaccine
Authors: Liu Pu , Jaffar Jade , Swisher Elizabeth , Hellstrom Karl Erik , Hellström Ingegerd
Published: February 2011

Background and objectives of the study: Many cancers, including ovarian cancer, over-express transforming growth factor-beta (TGF-ß), which helps them evade immune control through a variety of mechanisms. We investigated whether the efficacy of tumor cells to induce an immune response, when used intact or as part of a dendritic cell (DC)-based vaccine can be enhanced by inhibiting their production of TGFß1 via anti-sense.

Methodology: The TGFß1 expression by two ovarian cancer cell lines was silenced by transfection with a non-replicating lentivirus shRNA. Autologous PBMC were co-cultivated, in the presense of IL-2, with Mitomycin-C (MMC) treated cancer cells or mature dendritic DC that had been pulsed with tumor lysates. The in vitro sensitized lymphoid cells were evaluated by ELISPOT for production of IFN-γ and by flow cytometry.

Preliminary data: Quanitative ELISA showed that TGFß1 production in the two ovarian cancer lines was signficantly inhibited by transfection with shRNA. In vitro sensitization of autologous PBMC with either tumor cells where the TGFß1 gene had been silenced or with DC that had been pulsed with homogenates of tumor cells with a silencd TGFß1 gene induced a stronger IFNγ release and increased the frequency of NK cells and DC while it decreased the population of Treg cells. We are presently investigating the anti-tumor response in a mouse melanoma model after vaccination with murine bone-marrow derived DC which have benn pulsed with TGFß silenced tumor lysates.

Conclusions: The production, by many tumors, of TGFß1 interferes with their ability to induce an anti-tumor response. This is true both when intact tumor cells and DC pulsed with tumor homogenate are used as immunogen.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-320.
Abstract LB-344: Senescence-related genes signature in high grade gliomas
Authors: Coppola Domenico , Balducci Lodovico , Chen Dung-Tsa , Brem Steven , Egan Kathy , Dalton William , Yeatman Timothy
Published: February 2011

Background: The prognosis of high grade gliomas (HGG) worsens with the age of the patients. We hypothesized that overexpression of senescence-related genes indicates a poorer prognosis.

Materials and Methods: Frozen tumor samples from 60 patients of different ages (32 to 88 years) with the histologic diagnosis of HGG were macrodissected and arrayed on a custom version of the Affymetrix HG-U133 + GeneChip. There were 29 events (deaths) with a median survival time of 20 months. A set of 14 known senescence-associated genes (SAGs) including CCL2, CCL7, CDKN1A, COPG, CSF2RB, CXCL1, ICAM1, IGFBP3, IL6, IL8, SAA4, TNFRSF11B, TNFSF11 and TP53 were the focus of the study (Campisi J. et al. PLoS Biology, 2008). An overall senescence score was generated using principal component analysis to reflect the combined effect of the senescence-associated gene signature. Pearson correlation and Cox proportional hazards regression were used to examine the association of each SAG and the senescence score with age at onset and overall patient survival. False discovery rate was used to adjust for multiple comparisons.

Results: We found an age-related gradient in the senescence score representation of the combined SAGs (r = 0.40; p=0.0014). Ten SAGs were significantly correlated with age (false discovery rate at 5%): IGFBP3 (r=0.44), ICAM1 (r=0.40), IL6 (r=0.38), CDKN1A (r=0.35), TNFSF11 (r=0.34), TP53 (r=0.34), CXCL1 (r=0.31), CCL7 (r=0.30), COPG (r=0.29), and CSF2RB (r=0.29). The SAG signature was also correlated with poor prognosis (p=0.0098; median survival: 13 and 28 months, respectively, for high versus low senescence score). Two SAGs were significantly associated with patient survival: IL6 (14 versus 38 months) and COPG (13 versus 38 months).

Conclusion: We report for the first time an SAG signature in HGG associated with age at onset and patient outcome. This signature composed of genes involved in cell cycle regulation, apoptosis, cell proliferation, and angiogenesis may define a more aggressive subtype of high-grade glioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-344.
Abstract LB-348: Identifying novel oncogenes in ovarian cancer using integrative genomics and gene-knock downs
Authors: Ramakrishna Manasa , Williams Louise H. , Bearfoot Jennifer L. , Boyle Samantha E. , Sridhar Anita , Davis Sally , Gorringe Kylie L. , Campbell Ian G.
Published: February 2011

Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. To expedite identification of ovarian oncogenes we have integrated matching gene copy number and gene expression data from a cohort of 68 primary epithelial ovarian cancers to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (>40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 41% of these differentially expressed probesets also showed a strong positive correlation (r≥0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 43 known protein coding genes showed a strong positive correlation between expression and copy number. We have verified previously known ovarian oncogenes such as ERBB2 and also identified novel potential oncogenic drivers such as PUF60 (SIAHBP1) and TPX2. Functional characterisation and mutation analysis of these novel oncogene candidates will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-348.
Abstract LB-350: MicroRNA-301a, elevated in pancreatic cancer, targets tumor suppressor CDKN1A/p21 blocking cellular senescence and promoting invasion of pancreatic epithelial cells
Authors: Wang Jin , Katayama Hiroshi , Leblanc Aimee B. , Frazier Marsha L. , Killary Ann M. , Sen Subrata
Published: February 2011

MicroRNAs (miR) are negative regulators of gene expression and have been implicated to play important roles in the carcinogenic processes. Expression of miR-301a is elevated in multiple solid tumors including pancreatic cancer, small cell lung cancer, breast and liver cancer. However, the targets and function of miR-301a remain unknown. Here, we report that miR-301a targets CDKN1A/p21 tumor suppressor gene transcript leading to down regulation of the protein. Luciferase reporter assay of p21 3′UTR construct revealed that p21 is a direct target of miR-301a. Over expression of miR-301a in immortalized non-tumorigenic pancreatic epithelial cells (HPNE and HPDE) by transient transfection of precursor miR-301a resulted in down-regulation of p21 protein level with accompanying accelerated cell proliferation and increase in invasive potential detected in matrigel assays. Conversely, knockdown of miR-301a by antigomir301a resulted in elevated levels of p21 protein in pancreatic ductal adenocarcinoma cells (Panc-1 and Aspc-1). These cells displayed accumulation in G1 cell cycle phase and induction of cellular senescence. The results suggest that the use of synthetic antigomir301a may be a promising approach to pancreatic cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-350.
Abstract LB-351: miR-221&222 regulate cell motility in glioma cell lines targeting the protein phosphates PTPμ
Authors: Quintavalle Cristina , Garofalo Michela , Romano Giulia , Iaboni Margherita , Zanca Ciro , Brenca Monica , Nuovo Gerald , Croce Carlo Maria , Condorelli Gerolama
Published: February 2011

Glioblastoma multiforme (GBM) is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, patients's survival remains poor. Different evidences indicate that microRNAs might play a fundamental role in tumorigenesis, cell proliferation, migration and apoptosis. Through a microarray analysis we identified different microRNAs upregulated in the tumorigenic glioma cell lines U87 compared with the non tumorigenic cell lines T98G. Among different microRNAs we focused our attention on miR221&222. By bioinformatic analysis we identified a binding site for these two miRs in the 3′UTR of the protein phosphatase PTPμ. Luciferase assay confirmed that miR-221 and 222 bind to the 3′UTR of PTPμ and impairs its mRNA translation. Previous studies indicated that PTPμ suppresses cell migration/invasion and is downregulated in glioma. Significantly, we found that miR 221&222 overexpression induced a downregulation of PTPμ as analyzed by both western blot and RT-PCR. Furthermore, miR222&221 induced an increase in cell migration/invasion in glioma cells. Accordingly, we found an inverse correlation between miR221&222 and PTPμ in glioma cancer samples, as analyzed by immunohistochemistry and in situ hybridization on a tissue array representing 46 glioma and normal brain tissues. In conclusion, our results suggest that microRNA 221&222 might regulate glioma tumorigenesis through the control of cell migration process.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-351.
Abstract LB-36: Antibody mediated neutralization of sema4D reduces tumor angiogenesis and growth In Vivo.
Authors: Jonason Alan , Fisher Terrance , Torno Sebold , Bussler Holm , Li Ji , Winter Laurie , Kirk Renee , Howell Alan , Caplan Jeff , Kenney Patrick , Reilly Christine , Scrivens Maria , Evans Elizabeth , Paris Mark , Watkins Raymond , Zauderer Maurice , Leonard John E. , Smith Ernest S.
Published: February 2011

It has been demonstrated that semaphorin 4D (SEMA4D; CD100) is a potent pro-angiogenic molecule. SEMA4D binding to its receptor plexin-B1 (PLXNB1) on endothelial cells transactivates c-MET and promotes formation of new blood vessels and tumor growth in vivo. SEMA4D is over-expressed in a wide array of tumor types, and is also produced by inflammatory cells present in the tumor microenvironment. It has recently been demonstrated that in an environment lacking SEMA4D, the ability of mouse breast cancer cells to originate tumor masses and metastases is severely impaired, and that SEMA4D produced by tumor associated macrophages is required for tumor angiogenesis and growth.

In addition to its effects on endothelial cells, SEMA4D binding to PLXNB1 on tumor cells results in MET transactivation and migration of tumor cells. It has been further reported that overexpression of PLXNB1 and MET in breast and ovarian cancers is a negative prognostic factor. Tumors co-expressing PLXNB1 and MET were characterized as having a higher grade and an increased frequency of metastases.

Collectively, these results suggest that expression of SEMA4D, either by tumor cells or by tumor associated macrophages, may represent a mechanism of tumor neovascularization, and that expression of the SEMA4D receptor in tumors may further induce tumor invasion, thereby enhancing their growth, survival, and metastatic potential.

Antibody neutralization of SEMA4D thus may represent a new therapeutic strategy for cancer treatment. We selected a high affinity human IgG4 antibody that is specific for both mouse and human SEMA4D, and utilized several in vitro functional assays to demonstrate that this antibody blocks SEMA4D - PLXNB 1 interactions. Using both transplanted and orthotopic tumor models we demonstrated that antibody mediated SEMA4D neutralization in vivo inhibits tumor growth and tumor angiogenesis. This human antibody is currently in IND-enabling toxicology testing and we anticipate the initiation of human clinical trials in 2H 2010.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-36.
Abstract LB-370: A placental growth factor variant unable to recognize VEGFR-1 inhibits VEGF-dependent tumor angiogenesis via heterodimerization
Authors: De Falco Sandro , Tarallo Valeria , Capasso Onofrio , Vesci Loredana , Esposito Maria Teresa , Pastore Lucio , Orlandi Augusto , Pisano Claudio
Published: February 2011

Angiogenesis is one of the crucial events for cancer development and growth. Two members of vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if co-expressed in the same cell, are both required for pathological angiogenesis. We generated a PlGF1 variant, named PlGF1-DE in which the residues Asp72 and Glu73 were substituted with Ala. PlGF1-DE is unable to bind and activate VEGFR-1 but can heterodimerize with VEGF (J. Biol. Chem., 279: 43929-43939, 2004).

Here we demonstrate that overexpression of PlGF1-DE variant in tumor cells by adenoviral delivery or stable transfection significantly reduced the production of VEGF homodimer via heterodimerization, leading to a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel size, vessel stabilization by smooth muscle cells and monocyte-macrophage infiltration. PlGF1-DE/VEGF heterodimer, differently from wild type heterodimer able to induce VEGFR-1 dimerization as well as VEGFR-1/R-2 heterodimerization and activation, failed to induce receptors phosphorylation demonstrating that co-expression of PlGF1-DE variant in VEGF-producing cells effectively sequestered active VEGF, biasing toward the generation of a non-functional heterodimer.

Conversely, overexpression of PlGF1wt, while reducing VEGF homodimer production comparably to PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, did not inhibit tumor growth and vessel density compared to controls, but induced an increase of vessel size, vessel stabilization and monocyte-macrophage infiltration.

The ability of PlGF and VEGF-A to heterodimerize represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a ‘dominant negative’ of VEGF molecule and is a new candidate for anti-angiogenic gene therapy in cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-370.