bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5465: Functional and structural characterization of the high-affinity EphA2-binding site of ephrinA1
Authors: Tome Carla M. Lema , Palma Enzo , Wykoski Jill , Debinski Waldemar
Published: February 2011

Glioblastoma multiforme (GBM), a high-grade astrocytoma, is the most common primary brain tumor in adults. GBM is a highly lethal cancer associated with neurological morbidity, and median survival of GBM patients is between 12 to 15 months. Previous work in our laboratory demonstrated that the EphA2 receptor, a tyrosine kinase protein receptor, but not its preferred ligand, ephrinA1, is over expressed in GBM specimens and cell lines, and its expression is linked to malignancy grade and patient survival. Binding of ephrinA1 to EphA2 results in receptor downregulation and internalization, which mediates reversal of malignant properties in GBM cells. Moreover, ectopic expression of ephrinA1 in GBM cells results in its release into the extracellular environment as a soluble, monomeric protein which mediates its effects in a paracrine manner. Thus, we have begun studying the structure-function relationship of ephrinA1 with the goal of developing an optimal soluble ligand for EphA2. The region selected was the GH loop of ephrinA1, a highly conserved region known to mediate high-affinity binding of ephrinA5 to EphA3 and very recently confirmed to mediate ephrinA1 to EphA2 binding. Alanine substitution mutants of all amino acids within the ephrinA1 GH loop (H-108FQRFTPFTLGKEFKE123G-OH) were generated by site-directed mutagenesis. Sequence-verified ephrinA1-mutant plasmids were then transfected into U-251 MG GBM cells. Individual clones were selected and high-expressing clones were further cultured. Mutant ephrinA1 expression in the media was verified by western blotting (WB). Conditioned media (CM) was assayed for ephrinA1 content using a sandwich ELISA. Standardized wild-type and mutant CM was used in subsequent experiments. Functional activity of each mutant was assessed by EphA2 down-regulation in U-251 cells. Alanine substitutions at positions P113 and T115 enhanced, while mutants of ephrinA1 at positions F111, T112, F114, L116, K118, E119, and F120 had a deleterious effect on EphA2 down-regulation when compared to that induced by wild type ephrinA1. In addition, those mutations that enhanced receptor down-regulation also caused dramatic changes in cell morphology in GBM cells within 15 min of treatment. Furthermore, treatment using P113A and T155A CM decreased phosphorylation of ERK within 30 min, indicative of suppression of intracellular signaling survival pathways. Being that ephrinA1 is a tumor-suppressing factor in GBM, finding its optimal genetically engineered variant(s) will have an important impact on the design and production of novel anti-tumor agents. These molecularly-targeted agents will represent a novel approach to GBM 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 5465.
Abstract 5484: Simultaneous targeting of EGFR and MET pathways using nanotechnology improves the treatment outcomes of photodynamic therapy for pancreatic cancer
Authors: Zheng Lei Z. , Rai Prakash , Mai Zhiming , Spring Bryan Q. , Hasan Tayyaba
Published: February 2011

Pancreatic Cancer (PanCa) is difficult to treat due to its inherent resistance to most therapies. First line chemotherapy only has a 5% response rate that yields a dismal prognosis of 6-month median survival and <4% 5-year survival. Combination therapies only showed modest benefits and there are still vital unmet needs for alternative treatment that improve overall patient survival.

Photodynamic Therapy (PDT), a light-regulated cytotoxic therapy, showed promise as a new treatment modality for PanCa which improved median survival from 6 to 9.5 months in a pilot clinical trial. This result triggered our preclinical development of PDT combination regimen targeting proliferative pathways to further enhance treatment outcome.

Using nanotechnology, we designed and synthesized nano-constructs co-encapsulating multiple therapeutic agents that can simultaneously target EGFR and MET-regulated proliferative pathways in combination with cytotoxic PDT treatment. We tested these nano-constructs in orthotopic pancreatic cancer models for treatment efficacy and our major findings include:

1) The nanoscale multidrug delivery system allows simultaneous targeting of EGFR and MET-regulated proliferative pathways and offered the best reduction of local tumor burden and metastatic burden to lymph nodes, lung and liver. With only one time of treatment, the local tumor burden is reduced to 10% of that of non-treated, and significant reductions of organ metastasis were also achieved.

2) The nanoscale multidrug delivery system delivers PDT agent and MET inhibitor PHA-665752 with higher payload and reduces their systemic toxicity. The EGFR inhibitor Cetuximab is delivered intracellularly into cancer cells, which targets both intracellular and cell surface EGFR. Intracellular delivery of Cetuximab increased treatment efficacy compared to naked Cetuximab.

We are continuing optimizing the scheduling of individual treatments to further improve treatment outcomes.

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 5484.
Abstract 5485: Modular nanotransporters: a chimeric multi-functional platform for drug delivery in vivo
Authors: Sobolev Alexander S. , Slastnikova Tatiana A. , Rosenkranz Andrey A. , Schiffelers Raymond M. , Zalutsky Michael R.
Published: February 2011

A major challenge in the development of specific and effective targeted treatments is the paradoxical situation: one needs to exploit surface molecular targets for achieving tumor selectivity and nevertheless needs to deliver the therapeutic inside the target cell, e.g. to the cell nucleus because subcellular targeting is generally required for maximizing the therapeutic effect.

We created modular nanotransporters (MNT) unifying these 2 contradictory features into one chimeric macromolecule consisting of:- (i) an internalizable ligand module, (ii) a module with nuclear localization sequence, (iii) an endosomolytic module, and (iv) a carrier module. The modules retained their functions within the MNT: they showed high-affinity interactions with target receptors and alpha/beta-importin dimers, and form holes in lipid bilayers at endosomal pH. Anti-cancer therapeutics (photosensitizers, alpha-particle emitting radionuclides) carried by MNT acquired 20-3000 times greater cytotoxicity and cell specificity if compared with free therapeutics.

Herein, we have demonstrated in vivo that MNT possess a unique combination of properties compared with other scaffolds for drug delivery such as antibodies, nanoparticles, and liposomes. The MNT can be freeze-dried and reconstituted without loss of activity. Different modules can be inserted into the MNT in order to impart them with a specific set of capabilities; the modules are interchangeable, so they can be adapted for different cell types and/or different subcellular targets. The MNT can be used for the delivery of many types of therapeutics; they demonstrate low toxicity and low induction of delayed type hypersensitivity in mice. The MNT accumulated in murine tumors with high tumor: non-tumor ratios and displayed preferential accumulation in the subcellular compartment of choice, e.g. the cell nucleus. And finally, anti-cancer therapeutics carried by MNT acquired significantly greater cytotoxicity and cell specificity compared with free therapeutics.

We think that the MNT represent a new platform for targeted drug delivery that could be applied to the treatment of aberrant cell populations characterized by up regulation of a receptor for which there is a known ligand.

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 5485.
Abstract 5487: A new generation of tumor targetable polymeric nanoparticles for cancer therapy and imaging
Authors: Kim Jong-Ho , Bae Sang Mun , Kim Kwangmeyung , Lee Byung-Heon , Park Rang-Woon , Kwon Ick Chan , Kim In-San
Published: February 2011

Despite the significant progress in the development of anticancer technology, there is still no common cure for patients with malignant diseases. Conventional anticancer treatments are nonspecific to target killing of tumor cells, and always lead to systemic toxicity, causing undesirable severe side effects such as hair loss, damages to liver, kidney, and bone marrow. In order to achieve therapeutic levels of drug at the tumor site without damaging healthy organs and tissues, it is important to recognize the physiological differences between diseased and normal sites. Targeting drug delivery system is attracting increasing interest in research field to remove severe side effects and to increase reorganization of physiological differences. Polymeric nanoparticles can accumulate and extravasate within tumor tissue since their prolonged circulation by enhanced permeation and retention (EPR) effect, passive targeting system, which is achieved due to disorganized vascularization and defective vascular architecture induced in rapidly growing cancers. Even though passive targeting system shows moderate tumor therapeutic efficacy, active targeting drug delivery system has recently gained considerable attention to improve specificity. Especially, ligand/receptor-medicated targeting has emerged as a novel paradigm in the active targeting. Here, we have investigated active targeted polymeric nanoparticles by conjugation of tumor-specific homing peptide, AP-1 peptide, with modified glycol chitosan nanoparticles. Selection of peptides which show preferential binding to the target can be achieved by biopanning phage-displayed peptide libraries. Phage display technique is a promising tool for selecting peptides or proteins with specific binding properties from a lot of variants. AP-1 peptide can bind with interleukin-4 receptor which expressed on surface of several human cancer cells like H226 and MDA-MB231. Developed peptide-guided targetable polymeric nanoparticles show more specific binding and uptake as well as better therapeutic efficacy than net polymeric nanoparticles, passive targeted nanoparticles, which were confirmed with flowcytometry, immunofluorescence, histology, immunohitochemistry studies and animal experiments. In addition, they are better candidate as an imaging probe for cancer imaging and diagnosis than net polymeric nanoparticles. In conclusion, AP-1 peptide-conjugated chitosan nanoparticles are effective candidate as drug carrier and imaging probe for cancer therapy and imaging since AP-1 peptide induces the improvement of cell binding and uptake of nanoparticles.

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 5487.
Abstract 5488: Merging structure-activity relationship and nanotechnology to re-engineer cisplatin
Authors: Paraskar Abhimanyu S. , Soni Shivani , Chaudhuri Padmaparna , Sengupta Shiladitya
Published: February 2011

The use of nanovectors holds the potential to revolutionize cancer chemotherapy by specifically targeting tumor. A number of polymeric nanovectors are currently in development or in clinics, and are dramatically altering the pharmacodynamics and pharmacokinetics profile of the active agent. However, most of these polymeric constructs decrease the potency of the conjugated active agent, relying on increased uptake into the tumor for the improved therapeutic index. Furthermore, the nanovector formulation of small molecules such as cisplatin, which is a first-line therapy for multiple cancers, has been a challenge. So there is an urgent need to design a nanoparticle which will release a drug at its target site without decreasing efficacy of cisplatin. In the present study, we synthesized cisplatin-poly-isobutylene-maleic acid modified with glucosamine conjugate, which self assembles into nanoparticles with unique size depending on cisplatin to polymer ratio. The poly-isobutylene-maleic acid modification with glucosamine, mimicking glycans, not only increases hydrophilicity but also gives unique structural facilitation to polymer backbone so it can hold cisplatin by caboxylato and coordinate bond. The release kinetics of these nanoparticles reveals that the cisplatin release is pH dependent, and is released faster in lower pH than neutral or basic condition. We found that the these nanoparticles were taken up by cancer cells within 6 hours and inhibited proliferation and induced apoptosis in lung and breast cancer cells in vitro. In an in vivo lung and breast cancer models, the nanoparticles reduce tumor growth significantly with reduced systemic toxicity as compared to free cisplatin. In conclusion we have demonstrated that rationally re-engineered cisplatin nanoparticles improved therapeutic index for lung, breast and transgenic ovarian murine cancer models, which can be harnessed in the clinical management of cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5488.
Abstract 5489: The IVECT™ method, a versatile micelle for the delivery of chemotherapeutic drugs
Authors: Rios-Doria Jonathan , Carie Adam , Cardoen Gregoire , Sill Kevin
Published: February 2011

The development of new therapeutic agents has dramatically improved the quality of life and survival rate of patients suffering from a variety of disorders. However, drug delivery innovations are required to further improve the success rate of these treatments because approximately 40% of the newly discovered, promising small molecule therapeutics have poor aqueous solubility and cannot be delivered systemically. Rationally-designed, nanoscopic drug carriers offer a promising approach to achieving these goals due to their inherent ability to overcome multiple biological barriers. Ideally, a drug delivery system will not only be stable to systemic administration, but also be versatile enough to encapsulate a diverse range of molecules leading to decreased developmental and regulatory hurdles. These goals were the driving force behind the development of Intezyne's IVECT™ Method, which is based on tri-block copolymer micelle technology; spherical nano-sized drug carriers formed by the assembly of amphiphilic block copolymers in water. This modular drug delivery platform can be tailored to accommodate a wide range of drugs, biologics or diagnostics, improve post-administration stability, vary the micelle size, control the release of the therapeutic, and target specific diseased cells based on active targeting strategies. The purpose of this study is to demonstrate encapsulation of a variety of chemotherapeutics utilizing one polymer system, and show that crosslinked micelles have improved pharmacokinetic and toxicology profiles compared to the free drugs in animal models. To this end, formulations of gemcitabine, paclitaxel, doxorubicin and SN-38 were prepared and characterized for nanoparticle size distribution by DLS and active drug weight loading by HPLC. All micelles were 30-80 nm in size with weight loadings of between 2 and 10 %. PK data from cannulated rats demonstrated significant improvement in the CMax and AUC for encapsulated crosslinked formulations, compared to the free drugs. Toxicity profiling in CD-1 mice demonstrated that the polymer alone was very safe, as an MTD was not reachable, and that the crosslinked encapsulated formulations were tolerated at significantly higher doses compared to the free drugs. In conclusion, we have demonstrated that Intezyne's IVECT™ Method is extremely versatile and results in significant improvements in the overall pharmacology of chemotherapeutics.

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 5489.
Abstract 5498: Prognostic tools to predict the efficacy of anticancer drug treatment targeting Chromatin DNA or enzymes acting on DNA
Authors: Nalabothula Narasimharao , Ross Douglas D. , Carrier France
Published: February 2011

There is currently no prognostic tool that can predict or monitor a cancer patient's response to anticancer drugs targeting chromatin DNA or enzymes acting on DNA. Cancer patients generally receive chemotherapy without the possibility to determine a priori whether the proposed regimen will be effective. We have developed a simple quantitative assay to measure the efficiency of Topoisomerase 2 (Topo2) inhibitors on genomic DNA (gDNA). The technique combines PCR-stop assay to real time PCR (RT-PCR). Prior reports have demonstrated that Topo2 poisons such as VP16 (etoposide) and Doxorubicin, induce prominent DNA cleavage in the P2 promoter of the c-myc gene. Therefore, we have optimized the design of primers complimentary to the sequence of Topo2 sites in the c-myc locus to suit the sensitivity of real time PCR detection. In PCR-Stop assay, the extent of locus-specific DNA damage sustained by the cells is inversely proportional to the levels of DNA amplification. The PCR-stop-RT-PCR assay was first carried out on gDNA isolated from normal and cancer cells that were either untreated or treated with a combination of Histone Deacetylase and Topo2 inhibitors. Our data indicate that the levels of DNA damage correlate with the levels of histone H2AX phosphorylation and are proportional to the efficiency of the drug combination as measured by survival assays. The potential clinical application of the assay was tested on PBMC of two leukemia patients treated with oral VorinostatSAHA (400 mg/day for three days). Our data indicate that Vorinostat increased the sensitivity of both patients PBMC to VP-16 by about 40%. One main advantage of this technique is that it uses small amounts of sample DNA (50-100 ng) to assess the efficiency of a particular drug or drug regimen. This assay could be used as a diagnostic tool for pre-clinical or clinical assessment of drug efficiency, evaluation of regimen suitability for a given patient and determination of optimal drug doses.

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 5498.
Abstract 5514: Targeted liposomes in cancer therapy
Authors: Mulamalla Hari Chandana R. , Lambros Maria P. , Huang Ying
Published: February 2011

The purpose of this project was to design a liposomal formulation which targets the SLC transporter. Daunorubicin, an anticancer drug, used as a model was encapsulated in the targeted liposomal formulation and the cellular uptake and cytotoxicity of targeted liposomal daunorubicin (TLD) as well as the mechanisms of cellular uptake were evaluated using the A549 lung cancer cell line. A quantity of 3×105/2ml cells of A549 were cultured in each well of 6 well plates in RPMI medium. The cells were treated with TLD, non targeted liposomal (NTL) daunorubicin and free, non-encapsulated daunorubicin containing 10µM and 5µM drug. They were incubated for 5-6 hours at 37oC and analyzed using flow cytometry and fluorescence imaging. The cytotoxicity of the formulations was determined using MTS assay. The IC50 was determined from the growth inhibition curves of cell viability. Furthermore, the mechanism of internalization of the TLD formulation was studied using inhibitors specific to each pathway of internalization. The results show that the cellular uptake TLD showed a significant increase of 2- fold compared to that of NTL and free drug for both concentrations (10µM and 5µM). These results were also consistent with the cytotoxicity study showing higher cytotoxicity of TLD compared to NTL and free drug, The IC50 of TDL showed a 2.3-fold and 3.4-fold decrease compared to NTL and free drug, respectively. Also the data showed decrease in the cellular uptake of TLD when the macropinocytosis pathway was inhibited. Depletion of cholesterol in the cellular wall also reduced the cellular uptake of the TLD formulation. The results indicate that our novel targeted liposomal formulation shows enhanced cellular uptake and cytotoxicity likely due to macropinocytosis.

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 5514.
Abstract 5515: Design and synthesis of releasable folate-drug conjugates possessing optimized carbohydrate-based spacer units
Authors: Vlahov Iontcho R. , Santhapuram Hari Krishna R. , Wang Yu , You Fei , Kleindl Paul J. , Fan Mingjin , Vaughn Jeremy F. , Han Spencer J. , Leamon Christopher P.
Published: February 2011

The spacer region of releasable folate-drug conjugates may play an important role in “tailoring” the properties of the molecules for targeted therapies by: 1) modulating the affinity toward the folate receptor, 2) providing desirable hydrophilicity, and 3) changing dramatically the biological properties. Herein we describe the design and synthesis of a variety of novel carbohydrate-based spacers mimicking a peptidic backbone. After carefully optimizing the chemical structure of the carbohydrate units, we incorporated such spacers in a folate-containing molecular systems using standard solid phase synthesis (SPS). The synthetic utility of this elegant and innovative approach was exemplified by creating novel releasable folate conjugates of single cytotoxic agents, e.g. Tubulysin-Folate conjugate (currently in preclinical development) and Vinblastine-Folate conjugate, (already in clinical development). Furthermore, using the same powerful method, we synthesized several constructs containing a rational combination of two drugs (e.g. Vinblastine and Doxorubicin) attached to a single folate-targeting ligand via a dual-headed releasable Spacer-Linker system.

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 5515.
Abstract 5526: Development of dithiocarbamate/metal complexes in Pluronic® micelles for the treatment of malignancies
Authors: Rudrapatna Venkatesh K. , Kosak Ken M. , Terrazas Moises C. , Kennedy Thomas P. , Shami Paul J.
Published: February 2011

Dithiocarbamate/metal complexes are potently cytotoxic towards malignant cells of different tissues of origin. We synthesized DETC/Cu, a chelate of diethylthiocarbamate (DETC) and copper (Cu2+). We formulated DETC/Cu in P123 Pluronic® micelles and studied its in vitro anti-cancer activity against the following human cancer cell lines: HL-60 (acute myeloid leukemia), PPC-1 (prostate cancer), HEP-3B (hepatocellular carcinoma) and DLD-1 (colon cancer). Controls were treated with empty P123 Pluronic® micelles. Cells were cultured with varying concentrations of the drug for 24 hrs, 48 hrs and 72 hrs upon which cell growth was measured using an MTS cell proliferation assay. DETC/Cu inhibited growth in vitro at submicromolar concentrations. The 50% growth inhibitory concentration (IC50) was 0.015 μM, 0.023 μM, 0.10 μM and 0.19 μM for HL-60, PPC-1, HEP-3B and DLD-1 cells, respectively. We also performed caspase activity assays at various time points and concentrations using the DETC/Cu. At concentrations of 0.25 μM, 0.5 μM and 1 μM DETC/Cu increased caspase 3 activity after three hours by 162, 265 and 318 percent over baseline, respectively. At the same concentrations and time point, DETC/Cu increased caspase 8 activation by 97, 97 and 82 percent over baseline, respectively. At the same concentrations and time point, DETC/Cu increased caspase 9 activity by 128,164, and 178 percent over baseline, respectively. These results demonstrate that DETC/Cu activates both the intrinsic and the extrinsic apoptosis pathways.

In another experiment, the effect of DETC/Cu chelates on leukemia cell growth in vivo was studied. NOD/SCID IL2Rγnull mice were implanted bilaterally in the flanks with HL-60 cells. When tumors became palpable, treatment was started with DETC/Cu formulated in P123 Pluronic® micelles and administered intravenously every other day at a dose of 2 μmol/kg. There were 4 mice in each treatment group (Pluronic® P123 micelle control and DETC/Cu in P123 micelles). The experiment was terminated at day 15. At that point 3 out of 4 control animals had died and the one remaining alive had tumor volumes that had exceeded 10% of initial body weight. Tumor volumes were decreased by 22 and 33% in DETC/Cu-treated animals as opposed to vehicle controls on days 12 and 15, respectively. At the time of termination of the experiment, none of the animals treated with DETC/Cu had died.

These results demonstrate that dithiocarbamate/metal complexes hold great promise for medicinal development as anti-cancer agents.

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 5526.
Abstract 5549: p53-independent radiosensitization of lung cancer cells by inhibition of mTOR pathway
Authors: Nagata Yoko , Tojo Takashi , Ota Ichiro , Ohnishi Ken , Takahashi Akihisa , Ohnishi Takeo , Taniguchi Shigeki
Published: February 2011

A high extent of activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and mutations in the p53 gene are involved in lung cancer therapeutic resistance. The mammalian target of rapamycin (mTOR) acts as a downstream effector for Akt and activation of the Akt/mTOR signal is a contributing factor to decreased radiation sensitivity. The aim of this study was to examine whether the effect of rapamycin on radiation sensitivity is affected by cellular p53 gene status in two human non-small cell lung cancer (NSCLC) cell lines with the same genetic background except for their p53 gene status (H1299/wtp53 and H1299/mp53). Cellular radiation sensitivity was evaluated with colony formation assays and apoptosis assays, using Hoechst33342 staining. Rapamycin synergistically enhanced the cytotoxicity of radiation regardless of p53 gene status, and promoted the induction of apoptosis. Moreover, the combined treatment augmented the cytostatic effects of radiation independently of cellular p53 gene status. In agreement with this result, cell cycle analysis by flow cytometry showed increased G1 arrest and suppression of progression to S phase in both cell lines. Western blotting revealed a prominent p53-independent down-regulation of the mTOR signal and pro-survival molecule, cyclin D1 after the combined treatment, suggesting that rapamycin can enhance the effect of radiation through the repression of pro-survival signals and the reduction in the apoptotic threshold. This study indicates that inhibition of the mTOR signal may be a promising strategy for radiosensitization with no relevance to p53 gene status from two aspects of cell lethality and cell growth depression.

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 5549.
Abstract 5554: The proteome analysis of human lung adenocarcinoma
Authors: Tei Keikou , Kakehashi Anna , Yamano Shotaro , Wei Min , Nishiyama Noritoshi , Wanibuchi Hideki
Published: February 2011

Lung cancer is the leading cause of cancer death in the worldwide. Despite advances in early diagnostic methods and treatment modarities, lung cancer is often diagnosed at an advanced stage and has a poor prognosis. There is a need for the development of new biomarkers for early detection, defining cancer risk, prognosis, and therapy targets. The purpose of this study was to investigate the molecular background of lung carcinogenesis and to search useful biomarkers for clinical treatment including early detection and prognosis prediction of lung cancer by performing proteome analysis of human lung adenocarcinoma. Subjects were cases with primary lung adenocarcinoma on which surgical resection was performed. Tumor and adjacent non-tumor tissue sample were collected and proteome analysis was carried out. All through the examined cases, 52 proteins were found overexpressed and 13 proteins localized in extracellular space were detected and these proteins were thought as potential biomarkers associated with lung adenocarcinoma. We noticed one protein of them and performed immunohistochemical staining, 94% of cases were expressed. We assessed the statistical significance of the associations between expression level of this protein and clinicopathologic variables, it was showed that this protein had possibility to be a novel prognosis prediction biomarker of lung adenocarcinoma. This study enables the indentification of biomarker candidates for novel tumor markers and therapetic targets that will eventually lead to early detection, prevention and treatment of cancer.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5554.
Abstract 5566: Phosphoproteomic profiling of Met / K-Ras signaling in colorectal cancer by label-free LTQ-Orbitrap mass spectrometry
Authors: Organ Shawna L. , Tong Jiefei , Taylor Paul , Navab Roya , St. Germain Jonathan , Moran Michael F. , Tsao Ming-Sound
Published: February 2011

Background: Colorectal cancer (CRC) is the second leading cause of death from cancer overall. Mutations in the K-ras oncogene are the most frequently observed mutations in CRC. Activation of receptor tyrosine kinases, including Met, is also common in CRC. Co-activation of both Met and K-Ras could result in preferential over-activation of specific downstream cascade(s). To address the potential importance of Met and K-Ras tandem activation, we use the DLD1 CRC cell line model. DLD1 cells contain one mutated K-ras allele, resulting in a constitutively active protein. These cells express a relatively low level of Met. A derivative of this cell line, DKO4, has its mutant K-ras allele inactivated by somatic recombination. Our lab has previously shown that overexpression of Met in DLD1 cells (DLD1-Met) enhances the tumorigenicity and metastatic potential of these cells. This did not occur in DKO4-Met cells, suggesting that activated K-Ras is necessary for these Met-mediated processes. We hypothesize that Met and K-Ras co-operate to activate novel tumourigenic and metastatic signaling pathways.

Methods: We undertook a label-free mass spectrometry (MS)-based proteomics examination of protein-phosphotyrosine (pY) in the DLD1 CRC model. DLD1-Met and DKO4-Met cells were enriched for pY-containing peptides and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. Peptide quantification was achieved by using both peptide ion spectral counting and normalized extracted ion current methods. To determine ligand-dependent vs. -independent differences in Met signaling, the parental DLD1 and DKO4 cells were stimulated with an HGF time course. Previous results in the lab show that DLD1 cells with activated K-Ras have prolonged MAPK signaling in response to HGF compared to DKO4 cells.

Results: 9 proteins were found to be differentially regulated in these DLD1-Met vs. DKO4-Met. Validation by Western blotting in the empty vector and Met-overexpressing lines confirmed MS findings, as well as uncovered some pY changes mediated by both Met and K-Ras. Results from temporal analyses of pY in response to HGF stimulation will be presented to address this occurrence and suggest mechanisms of Met and K-Ras dependent tumourigenesis.

Conclusions: We have identified a phosphoproteomic network associated with Met/K-Ras signaling. It may include novel therapeutic targets or prognostic markers for CRC.

Supported by the CIHR MOP-64345 and the CIHR Banting and Best Doctoral Research Graduate Scholarship to SO

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 5566.
Abstract 5569: N-glycosylation in cultured cells and hepatocellular carcinoma
Authors: Brnakova Zuzana , Chandler Kevin B. , Bekesova Slavka , Lenco Juraj , Bebu Ionut , Kosti Ourania , Shetty Kirti , Korba Brent E. , Ressom Habtom W. , Varghese Rency S. , Novotny Milos V. , Goldman Radoslav
Published: February 2011

Purpose: Incidence of hepatocellular cancer (HCC) continues to increase primarily due to hepatitis C viral (HCV) infection. Prognosis and survival of patients is highly affected by the disease stage at the time of diagnosis. Our mass spectrometric study evaluated N-glycans during the progression of HCV infection to cancer. Glycosylation of immunoglobulins and other serum proteins was examined in cultured cells and in serum of patients with hepatocellular carcinoma. Methods and Results: Protein associated N-glycans were released with PNGaseF and analyzed by MALDI-TOF/TOF analysis following solid phase permethylation. Analysis of less than 0.04 ml of serum led to relative quantification of 70 N-glycan structures. Immunoaffinity isolation of immunoglobulins and other serum proteins allowed us to study protein-specific glycosylation. Progression of HCV infection to HCC was strongly associated with changes in the glycosylation of immunoglobulins. In a pilot case-control study (25 HCC cases and 35 controls), N-glycosylation of eight glycans was significantly different in HCC compared to chronic liver disease controls. The glycosylation of serum proteins was further compared to the proteins secreted by the Huh-7.5 cell line. Conclusion: This study demonstrates mass spectrometric analysis of 70 N-glycans in serum, cultured cells, and isolated proteins. Our results show that the analysis of permethylated N-glycans helps to define changes associated with the progression of HCV infection to HCC. Evaluation of glycan abundance suggests the potential to use glycans for the detection of liver disease.

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 5569.
Abstract 557: Galectin-3 binding protein produced by neuroblastoma cells stimulates the expression of interleukin-6 in bone marrow mesenchymal cells
Authors: Silverman Ayaka M. , Fukaya Yasushi , Shimada Hiroyuki , Groshen Susan G. , DeClerck Yves A.
Published: February 2011

There is recent evidence that bone marrow-derived mesenchymal cells (BMMC) play an important role in bone metastasis in several cancers, including myeloma and neuroblastoma. We previously reported that in neuroblastoma these cells are a source of interleukin-6 (IL-6) that promotes the growth and survival of neuroblastoma cells and bone degradation. We have recently isolated and identified Galectin-3 binding protein (Gal-3BP) as a soluble factor produced by a neuroblastoma cell line as a factor upregulating the production of IL-6 by BMMC. Here we document the production of Gal-3BP in several neuroblastoma cell lines, in particular those without MYCN amplification that correlates with their ability to induce the production of IL-6 in BMMC. The stimulation of IL-6 by Gal-3BP is mediated by Gal-3, a multifunctional glycoprotein that binds Gal-3BP and is present in BMMC. It is also dependent on the activation of the Ras/Raf/MEK/ERK1/2 pathway by Gal-3BP/Gal-3 and is blocked by the ERK1/2 inhibitor PD980559. Transcriptional activation of IL-6 by Gal-3BP/Gal-3 requires a 115 bp region (−97 to −212) in the IL-6 promoter that contains recognition motifs for a cAMP responsive element binding protein (CREB) and a CCAAT/enhancer binding protein (C/EBP). The expression of Gal-3BP was also examined by immunohistochemistry in 38 primary neuroblastoma tumors with (n=9) and without (n=29) MYCN amplification. This analysis revealed that Gal-3BP is paradoxically more abundant in MYCN non-amplified tumors. This unanticipated observation suggests a significant difference in the Gal-3BP/Gal-3/IL-6 pathway between subgroups of neuroblastoma tumors.

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 557.
Abstract 5572: Analysis of changes in RTK phosphorylation using microplate-based antibody arrays
Authors: Finkel David J. , Johnson Wade M. , Johnson Wade M. , Buehl Christopher J. , Coleman Rebecca A. , Campos Maria T. , Luhowskyj Stefan M. , Boldin Kristina M. , Stoesz Steven P. , Campos-Gonzalez Roberto , Krzyzek Richard A. , Brumbaugh Kathryn M.
Published: February 2011

Receptor-tyrosine kinases (RTKs) are transmembrane proteins that have been implicated in various cancers and are considered therapeutic targets. The phosphorylation of RTKs on tyrosine residues leads to their activation. Therefore, methods to identify the effect of RTK inhibitors or modifying antibodies on RTK phosphorylation are effective tools for drug screening. The Proteome Profiler 96 Human Phospho-RTK Antibody Array is a unique and powerful, plate-based multiplex immunoassay that utilizes ELISA techniques to measure changes in the phosphorylation of multiple RTKs simultaneously. In this assay, antibody/antigen reactions take place on the surface of a microplate that has been pre-spotted with antibodies against each RTK. Each spot corresponds to a unique analyte. A horseradish peroxidase-conjugated anti-Phospho-Tyrosine detection antibody is subsequently added to each well. A chemiluminescent substrate mix and a camera imaging system are used to determine the relative amount of analyte bound in individual spots. Experiments were performed by incubating the MDA-MB-453 breast cancer cell line with specific kinase inhibitors or with antibodies to ErbB receptors prior to NRG1-

β1/HRG1-

β1 treatment. Cells were lysed directly in 96-well plates and transferred to the Proteome Profiler 96 Human Phospho-RTK Array for analysis. NRG1-

β1/HRG1-

β1-dependent tyrosine phosphorylation of all four ErbB receptors was monitored simultaneously and the effects of different kinase inhibitors or modifying antibodies were determined. Proteome Profiler 96 Antibody Arrays offer the advantages of small sample and volume size requirements with as little as 5

μg of total cellular protein in a total volume of 100

μL being required, while still offering the specificity and sensitivity of sandwich immunoassays.

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 5572.
Abstract 5575: Mass spectrometry analysis of the posttranslational modifications of alpha-enolase from pancreatic ductal adenocarcinoma cells
Authors: Zhou Weidong , Capello Michela , Fredolini Claudia , Novelli Francesco , Liotta Lance A. , Petricoin Emanuel F.
Published: February 2011

Enolase is one of the most abundantly expressed cytosolic proteins in many organisms, and it is a key glycolytic enzyme that catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Recently, accumulating evidence revealed that, in addition to its innate glycolytic function, the well characterized old enzyme enolase is a multifunctional protein and plays an important role in several biological and pathophysiological processes: 1) by using an alternative start codon, the α-enolase mRNA can be translated into a 37 kDa protein which lacks the first 96 amino acid residues. This protein is localized in the nucleus and can bind to the c-myc P2 promoter and negatively regulate transcription of the proto-oncogene; 2) α-enolase has been detected on the surface of hematopoietic cells such as monocytes, T cells and B cells, neuronal cells and endothelial cells as a strong plasminogen-binding receptor, modulating the pericellular and intravascular fibrinolytic system. The expression of α-enolase on the surface of a variety of eukaryotic cells has been found to be dependent on the pathophysiological conditions of these cells, however, how α-enolase is displayed on the cell surface remains unknown; 3) increased expression of enolase has been reported to correlated with progression of tumors, such as neuroendocrine tumors, neuroblastoma and lung cancers, and enolase has been considered to be a diagnostic marker for many tumors. Our previous studies demonstrated that α-enolase is up-regulated at both the mRNA and protein levels in pancreatic ductal adenocarcinoma (PDAC). PDAC is the fourth leading cause of cancer-related deaths in Western countries. The mean life expectancy is 15-18 months for patients with local and regional disease, and only 3-6 months for those with metastatic disease. In this present work, we further characterized the α-enolase from PDAC by reversed-phase liquid chromatography nanospray tandem mass spectrometry (LC-MS/MS) analysis and an LTQ-Orbitrap instrument, and identified multiple posttranslational modifications of α-enolase, such as phosphorylation, acetylation, and methylation. The mass spectrometer LTQ-Orbitrap provides high accuracy mass measurement that is essential for the validation of modified peptide identifications and the reduction of false positive identifications.

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 5575.
Abstract 5578: Quantitative kinase inhibitor profiling in native cell and tissue extracts
Authors: Patricelli Matthew P. , Nomanbhoy Tyzoon K. , Kozarich John W. , Gray Nathanael S. , Zhang Jianming , Wu Jiangyue
Published: February 2011

Protein kinases are currently the subject of intense pharmaceutical industry attention. An array of assays and tools have been developed to study their biological and structural properties, however despite these efforts, many questions remain regarding the nature and functional consequences of kinase regulatory events in vivo. We previously detailed a probe-based method for comprehensively probing the ATP binding sites of native protein kinases directly in cell or tissue lysates. Here we report several advances that dramatically increase the sensitivity, quantitative accuracy, and efficiency of this method. Using these improved methods, we determined the Kd values of ATP, Staurosporine and several clinically relevant kinase inhibitors against more than 200 protein and lipid kinases in two cell lines (HL60 and PC3). While the data supports many general trends and confirmed targets of the inhibitors identified through recombinant kinase assays, several striking differences were found. Detailed investigation of these differences reveal that native kinases can exhibit dramatically different behavior when compared to their recombinant counterparts. Overall these data provide an unprecedented picture of native kinase behavior and have profound implications for the development of kinase inhibitors.

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 5578.
Abstract 5582: NY-ESO-1-specific T-cells in melanoma patients with good clinical outcome after vaccination using mRNA as the immunogen
Authors: Zelba Henning , Weide Benjamin , Garbe Claus , Pawelec Graham P. , Derhovanessian Evelyna
Published: February 2011

Although success in active cancer immunotherapy is often disappointing, vaccination against cancer- associated antigens has become an important tool in cancer management, resulting in an increasing list of possible antigen targets. However, previous studies have shown that vaccination against certain antigens results in a better clinical outcome than others. This indicates the importance of immune monitoring by helping to choose the antigens with the highest potential for success.

In order to address this issue, we have analysed CD4+ and CD8+ T-cell responses against four common melanoma-associated antigens, namely NY-ESO-1, Melan-A, MAGE-A3 and Survivin, in melanoma patients undergoing immunotherapy. Blood samples from 5 long-term survivors after treatment with anti-CTLA-4 antibody as well as 12 patients vaccinated with mRNA encoding these four antigens were tested in this study. Antigen-specific T-cell responses were detected using peptide mixtures spanning the whole sequence of the antigen after one round of in vitro sensitization. Intracellular production of six different cytokines (IFN-γ, TNF, IL-2, IL-4, IL-10 and IL-17) was measured in CD4+ and CD8+ T-cells by 14 colour multiparameter flow cytometry, allowing simultaneous analysis of the phenotype and effector functions (TH1, TH2, TH17) on a single-cell basis.

NY-ESO-1-specific CD4+ and/or CD8+ T-cell responses were detected in 4/5 long-term (up to 3 years and continuing) survivors receiving anti-CTLA-4 treatment, whereas responses against the remaining three antigens were either absent or present at lower frequencies. In the mRNA vaccination trial, NY-ESO-1-specific responses were detected in the only stage IV patient with >25% shrinkage in the size of the metastasis and two stage IV patients, who have remained tumor-free for 21 and 12 months since start of the vaccination. NY-ESO-1-specific immune responses were present in only 1/9 patients who progressed or died during this period. In all 6 patients with clinical benefit, the NY-ESO-1-specific T-cells produced high levels of IFNγ and TNF.

Our data suggest that NY-ESO-1 might be a promising target antigen in melanoma 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 5582.