bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B260: Antitumor activity of HM781-36B, an irreversible pan-HER inhibitor, alone or in combination with cytotoxic chemotherapeutic agents in gastric cancer cells
Authors: Nam Hyun-Jin , Kim Hwang-Phill , Yoon Young-Kwang , Hur Hyung-Seok , Kim Maeng-Sup , Lee Gwan-Sun , Han Sae-Won , Oh Do-Youn , Im Seock-Ah , Kim Tae-You , Bang Yung-Jue
Published: June 2012

Background: HM781-36B is a novel quinazoline-based irreversible pan-HER inhibitor. Although the effects of many other pan-HER inhibitors (CI-1033, PF00299804, BMS-599626, BMS-690514 and JNJ-28871063) have been studied in various types of cancer, data regarding the use of pan-HER inhibitors to treat gastric cancers are limited. In this study, we evaluated the therapeutic potential of HM781-36B for the treatment of gastric cancers by testing its in vitro and in vivo efficacy. In addition, we further characterized its activity when administered in combination with chemotherapeutic agents.

Methods: Experiments were conducted in gastric cancer cell lines. Enzyme activity assay was conducted to determine the IC50 values of HM781-36B for kinase inhibition and MTT assay was used for determining growth inhibitory effect. Cell cycle distribution was analyzed by flow cytometry and apoptotic effect was measured by annexin V assay. And N87 xenograft model was used to see tumor regression effect. Western blot analysis was used to verify mechanism of this compound.

Results: We found that HM781-36B suppressed the proliferation of HER2-amplified gastric cancer cell lines (SNU216 and N87) potently when compared to the effects of other HER family tyrosine kinase inhibitors (gefitinib, lapatinib, BIBW-2992 and CI-1033). Furthermore, we found that HM781-36B inhibited the phosphorylation of HER family and their downstream signaling proteins, which resulted in apoptosis through the caspase-mediated mitochondrial pathway and G1 arrest. The apoptotic process requires the induction of the proapoptotic BH3-only BCL2 family member, BIM, and HM781-36B led to a dramatic increase in the BIM level in SNU216 and N87 cells. Consistent with these in vitro findings, HM781-36B showed exceptional anti-tumor activity in a xenograft model of N87 cells. Furthermore, HM781-36B exerted synergistic effects when administered in combination with 5-fluorouracil or cisplatin in both HER2 amplified cells and HER2 non-amplified gastric cancer cells.

Conclusion: Taken together, these data suggest that HM781-36B may be useful as a targeted therapy for the treatment of HER2 amplified gastric cancer as well as a chemosensitizer of cytotoxic chemotherapeutic agents used to treat gastric cancer, even in HER2 non-amplified gastric cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B260.
Abstract B262: Tyrosine kinase inhibitors induce rapid cellular alterations in breast cancer cells in vitro
Authors: Silvia Carloni , Fabbri Francesco , Brigliadori Giovanni , Ulivi Paola , Amadori Dino , Silvestrini Rosella , Zoli Wainer
Published: June 2012

Purpose: Alterations in tyrosine kinase (TK) expression or functionality have been linked to tumor growth, and the unraveling of TK pathways has led to the discovery of novel anticancer drugs. The aims of this study were to evaluate cytotoxic and multidrug resistance mechanisms, such as Ca2+ homeostasis and efflux pump activity, of three tyrosine kinase inhibitors (TKIs), lapatinib, sorafenib and gefitinib, in two breast cancer cell lines.

Methods: Two human breast cancer cell lines, Estrogen Receptor (ER) positive MCF-7 and ER negative BRC-230 were exposed to the investigated drugs for 20, 40, 60, 120 and 240 min. Cytotoxicity, cytosolic Ca2+ levels, extrusion pump activity and mitochondrial membrane depolarization were assessed by flow cytometry. Expression of drug efflux genes was determined by RT-PCR, and protein expression of drug targets by Western blot.

Results: MCF-7 cells did not express EGFR, p-EGFR, Her-2 or p-Her-2, while BRC-230 were positive for EGFR and p-EGFR only. ERK1/2 was equally expressed in both cell lines, but only BRC-230 were positive for p-ERK1/2. MEK and p-MEK were expressed in both cell lines, albeit more intensively in BRC-230. As expected, based on their drug target expression profiles, gefitinib and lapatinib were found to induce cytocidal effects and mitochondrial membrane depolarization in BRC-230 only, while sorafenib caused apoptosis and loss of mitochondrial potential in both cell lines. All three drugs triggered a rapid and biphasic increase in cytosolic [Ca2+] (after a 20 min exposure), and a decline in xenobiotic extrusion pump activity that was independent of efflux gene downregulation. The thapsigargin-induced depletion of endoplasmic reticulum Ca2+ blocked the increase in cytosolic Ca2+, but had no effect on multidrug transporter activity or on mitochondrial membrane depolarization.

Conclusions: Gefitinib, lapatinib and sorafenib induced a rapid increase in cytosolic [Ca2+] and mitochondrial membrane depolarization, in addition to blocking drug transporter activity, independently of the expression of specific drug targets and of drug-induced cytotoxicity. These data suggest that TKIs may elicit different molecular mechanisms to those previously reported in the literature. Further research of these pathways is warranted in order to identify new, more effective strategies for cancer treatment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B262.
Abstract B264: IC261 induces cell cycle arrest and apoptosis of human cancer cells via a CK1δ/ε independent mechanism
Authors: Cheong Jit Kong , Virshup David M.
Published: June 2012

Casein kinase 1 delta (CK1δ) and CK1 epsilon (CK1ε) regulate Wnt/β catenin signaling and their dysregulation has been implicated in a variety of human cancers. Kinome RNAi screens and as well as targeted studies have identified CK1δ and CK1ε as prosurvival factors in a variety of cancer cell lines. While RNAi knockdown of CK1δ or CK1ε displayed only modest growth inhibition of various cell lines, the CKIδ/ε selective inhibitor 3-[2,4,6-(trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261)(with an IC50 in the 20–50 µM range) has proven even more effective at selective killing of cancer cells, even at submicromolar levels. This finding has been taken as supportive evidence for the role of CKIε and CKIδ in cancer. The recent development of a selective and nanomolar CKIδ/ε inhibitor, PF670462, affords an opportunity to further explore this hypothesis. We found that PF670462 effectively blocked CK1 kinase activity and suppressed Wnt/β-catenin signaling in both HEK293 cells and HT1080 fibrosarcoma cells at submicromolar concentrations. However, consistent with the results of siRNA knockdown of CK1ε, PF670462 failed to induce cancer cell death at these concentrations. In contrast, we found that while 100 nM IC261 did not inhibit of CK1ε activity nor block Wnt/ catenin signaling in vivo, it resulted in the rapid induction of G2/M arrest of cell cycle and apoptosis in human cancer, but not non-transformed, cells. Taken together, inactivation of CK1δ/ε and subsequent block of Wnt/β catenin signaling may not be sufficient to inhibit cancer cell survival. Furthermore, IC261 potently regulates additional cancer cell targets at concentrations much lower than that required for CK1 inhibition and these novel unknown targets appear to be important for the maintenance of human cancer cell survival. Identification of the IC261 targets may provide insights into the mechanism of selective cancer cell toxicity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B264.
Abstract B267: AR-mTOR-1: A potent, selective mTORC 1/2 kinase inhibitor for the treatment of malignancy
Authors: Wallace Eli M. , Xu Rui , Josey John , Gross Stefan D. , DeMeese Lisa , Fischer John , Miknis Greg , Humphries Mike , Regal Kelly , Fell Brad , Burkard Michael , DeWolf Walt E. , Gloor Susan , Hastings Gregg , Zuzack John , Winkler James , Koch Kevin
Published: June 2012

Through alterations in the PTEN and PI3K genes, the PI3K / Akt pathway is constitutively activated in human cancers. mTOR kinase plays an unique role in this pathway as the key component of two independent signaling complexes (mTORC1 (raptor - rapamycin sensitive) and mTORC2 (rictor - rapamycin insensitive)) that are involved at multiple branch points in this signaling cascade. As such, inhibition of mTOR kinase inactivates both mTOR complexes and therefore serves as an attractive means to target this integral pathway for the treatment of human malignancy.

We report the biological and pharmaceutical evaluation of our selective mTOR 1/2 kinase inhibitor AR-mTOR-1. AR-mTOR-1 inhibits mTOR kinase with an IC50 of < 10 nM while maintaining selectivity against PI3K as well as a panel of additional lipid kinases, serine/threonine kinases and cytoplasmic and receptor tyrosine kinases. In mechanistic cellular assays, AR-mTOR-1 inhibits pAkt (Ser473), 4E-BP1 (Thr36/46) and pS6 (Ser235/6) with nanomolar potency, thus demonstrating inhibition of signaling from both mTORC1 and mTORC2 complexes. In line with its enzymatic selectivity over PI3K , AR-mTOR-1 does not significantly inhibit pAkt (Thr308) in cells. AR-mTOR-1 is broadly anti-proliferative in both epithelial and hematologic cancer cell lines, irrespective of mutational status, with IC50's ranging from 30 to 550 nM across 20 cell lines, suggesting the potential for broad clinical activity. Once daily dosing of AR-mTOR-1 in several mouse xenograft models, including PC3 prostate, U87 glioblastma, and H460 lung, results in robust anti-tumor activity. Finally, AR-mTOR-1 possesses desired in vitro and in vivo preclinical ADME properties including low clearance, high permeability and good absorption in three preclinical species. In total these data demonstrate that selectively targeting mTORC1 and mTORC2 with AR-mTOR-1 holds promise for broad spectrum clinical utility as a single agent across a wide array of cancer types.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B267.
Abstract B31: Multicellular resistance to bortezomib acquired by mesothelioma spheroids can be reversed by modulation of the Bcl-2 repertoire
Authors: Barbone Dario , Fennell Dean A. , Broaddus V. Courtney
Published: June 2012

Whereas cancer cells may attain a chemoresistant phenotype on a single cell basis (unicellular resistance), they acquire further resistance to apoptosis when they form three-dimensional (3D) structures (multicellular resistance). The multicellular spheroid, a type of in vitro 3D cell culture, has emerged as a valuable platform to study the acquired multicellular resistance of cancer. We have previously shown that mesothelioma, a recalcitrant tumor with significant apoptotic resistance, manifests a high degree of multicellular resistance in vitro when grown as 3D spheroids. One of the treatments considered for mesothelioma is bortezomib, a proteasome inhibitor; bortezomib has been effective as a single agent in multiple myeloma but has been generally ineffective in solid tumors. Bortezomib has been shown to modulate several pro-/anti-apoptotic members of the Bcl-2 family, including upregulation of pro-apoptotic Noxa, thereby shifting the balance towards apoptosis. We asked whether mesothelioma spheroids develop multicellular resistance to bortezomib and whether changes in the Bcl-2 repertoire could account for the acquired resistance. Using monolayers and spheroids grown from two human mesothelioma cell lines, M28 and REN, we found that these cell lines acquired significant multicellular apoptotic resistance to bortezomib when grown as spheroids. Nonetheless, bortezomib inhibited proteasomal activity equally in both monolayers and spheroids and caused the accumulation of a comparable amount of ubiquitinated proteins in both systems. Despite these similarities, we found that the Bcl-2 repertoire was different in spheroids compared to monolayers, both at baseline and after bortezomib. One major difference was that Noxa, although upregulated in monolayers by bortezomib as expected, was not expressed in the spheroids, either at baseline or after bortezomib. The lack of Noxa accounted for some of the resistance because we found that a cell-permeable Noxa-BH3 peptide partially restored the apoptotic response to bortezomib in 3D spheroids. ABT-737, an inhibitor of the anti-apoptotic proteins Bcl-3/xL/w, was even more effective at reversing bortezomib resistance acquired by cells in 3D. In fact, when given alone, ABT-737 was selectively toxic for the spheroids, inducing apoptosis by itself in 3D spheroids but not in the 2D monolayers. In conclusion, we find that Bcl-2 proteins can account for apoptotic resistance in 3D. Manipulation of the Bcl-2 repertoire may be effective alone and may also improve the therapeutic response of mesothelioma to bortezomib therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B31.
Abstract B39: CXCL1 as a putative tumor marker for gastric cancer progression
Authors: Jung Minkyu , Jung Jae-Joon , Noh Se Won , Jeung Hei-Cheul , Kim Tae Soo , Kim Hyo Song , Noh Sung Hoon , Roh Jaekyung , Chung Hyun Cheol , Rha Sung Young
Published: June 2012

Gastric cancer (GC) is a heterogeneous disease and current tumor markers are limited in its application. Therefore finding molecular markers that are able to predict the potential of tumor progression is important for appropriate individualized therapy. With the Cancer Metastasis Research Center (CMRC) microarray database (17K cDNA microarray), we identified differentially expressed genes between 96 cancer and 98 normal gastric tissues using Significant Analysis of Microarray (SAM). Among them, more than 2-fold overexpressed genes in tumor tissues which encode secreted proteins were selected. The selected gene was validated with ELISA assay using sera of 96 GC patients and 48 healthy donors. We first selected 6510 genes which were differentially expressed between 96 cancer and 98 normal gastric tissues with minimal false discovery rate (FDR) of 0.005%. Then, out of 6510 genes, we picked 386 genes which encode secreted proteins based on SOURCE database. Among 386 genes, we focused on the 55 genes overexpressed more that 2-fold in GC compared to normal. With Ingenuity Pathway Analysis, we found 34 genes related to cancer. Especially, CXCL1, chemokine growth-regulated oncogene 1, has been known for its relation with cancer progression in various cancer type, but GC. CXCL1 levels in serum samples of GC patients were significantly elevated compared with healthy donors (p<0.05). Within GC patients, CXCL1 serum levels were increased according to the tumor stage and lymph node metastasis. These findings provide that CXCL1 could be a candidate marker for gastric cancer progression.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B39.
Abstract B50: Leptin and leptin receptor expression in breast cancer
Authors: Yom Cha Kyong , Kim Jun Ho , Kim Hee Sung , Park Yong Lai
Published: June 2012

Background: Leptin is a multifunctional hormone produced by the adipose tissue and involved in the regulation of food intake and energy balance. The aims of this study is to evaluate leptin and leptin receptor (Ob-R) expression in human breast cancer and determine whether it influence the prognosis of the patients with breast cancers.

Methods: We examined the correlation for leptin and Ob-R expression and breast cancer-related pathobiological markers by immunohistochemistry in five hundred and seventeen patients with breast cancer. We analyzed leptin and leptin receptor (Ob-R) expression for overall survival (OS) and relapse-free survival (RFS).

Results: Positive cytoplasmic immunoreactivity was found in 39% for leptin and 79% for Ob-R. The expression of leptin in breast cancer correlated with high Ki-67 labeling index (p=0.019). The leptin expression showed significant correlation with triple negative (TN) type (p=0.022) and epidermal growth factor receptor (EGFR) reactivity (p=0.05). The frequency of Ob-R expression was higher in non-TN breast cancers (p=0.045) and the breast cancers with high Ki-67 labeling index (p=0.06). In univariate survival analysis, clinicopathologic variables with prognostic value included histologic grade, T stage, N stage, HER2 status, Bcl-2, p53, and Ki-67 expression (p<0.05). The patients with leptin-positive breast cancers showed longer OS in negative hormone receptor status. In multivariate survival analysis with Cox proportional hazards model, leptin expression was independent prognostic marker (Hazard ratio=0.22, 95% CI 0.06 and 0.79, p=0.02) in hormone receptor negative breast cancers.

Conclusion: We conclude that leptin leptin expression in breast cancers is significantly associated with Ki-67 labeling index, suggesting association with proliferation activity. In addition, leptin expression is a indicator of favorable tumor features and better survival of hormone receptor negative breast cancer patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B50.
Abstract B63: Apaf-1 mislocalization is a strong indicator of drug resistance in B cell lymphomas
Authors: Hirpara Jayshree L. , Loh Thomas , Goh Boon Cher , Pervaiz Shazib
Published: June 2012

Resistance to chemotherapy remains a challenge in the clinical management of diffuse B cell lymphomas despite aggressive chemotherapy such as CHOP and monoclonal CD20. We previously reported that sequestration of Apaf-1 to membrane lipid rafts was responsible for apoptosis resistance in B-cell lymphoma cell lines1. Here, we extended our studies to 60 clinical biopsies from patients with B-cell lymphomas, T-cell lymphomas and reactive lymphadenopathy, to investigate if the resistance to drug-induced apoptosis was, indeed, a function of Apaf-1 mislocalization. Firstly, cells were separated from these biopsies and their sensitivity to a variety of apoptosis inducing agents was assessed. Whereas, most T cell lymphomas as well as reactive lymphadenopathy cells were sensitive to apoptotic stimuli, B cell lymphomas exhibited strong resistance. We then investigated the expression of Apaf-1 and its intracellular localization in these clinical biopsies. To do so, cell fraction was performed to separate cytosol from membrane-enriched fractions. The latter were further subjected to density gradient centrifugation to obtain lipid raft fractions. We show that Apaf-1 was expressed in total cell lysates from B and T cell lymphomas, however upon fractionation the localization was strikingly different. In T cell lymphoma samples as well as in cells derived from reactive lymphadenopathy biopsies, Apaf-1 expression was prominently detected in the cytosol, which correlated with the sensitivity of the cells to apoptotic stimuli. In contrast, whereas cytosolic Apaf-1 expression was significantly lower or absent in almost all B cell lymphomas analyzed, increased localization of the protien was detected in membrane lipid rafts. The latter was confirmed by immunohistochemical analysis of tissues from the same biopsy specimens. Interestingly, the resistance of B cell lymphomas to apoptotic execution (drug-induced or death receptor-mediated) was significantly bypassed upon incubation of cells with pharmacological agents that facilitated the dissociation of Apaf-1 from the lipid rafts to the cytosol. Taken together, our results implicate Apaf-1 mislocalization as a potential diagnostic marker for B-cell lymphomas as well as a predictor of response to therapeutic management.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B63.
Abstract B83: Modulation of the HSP90 cochaperone carboxy-terminus HSC70-interacting protein (CHIP) increases cellular sensitivity to heat shock protein 90 (HSP90) inhibition
Authors: Hughes Edward G. , Sharp Swee Y. , Workman Paul
Published: June 2012

HSP90 operates with cochaperones to enable the correct folding, stabilisation and functioning of multiple oncogenic protein kinases, hormone receptors, and transcription factors, thus making HSP90 an exciting drug target. HSP90 inhibition results in proteasomal degradation of client proteins via ubiquitination by E3 ligases such as CHIP. This cochaperone functions with HSP90 as a molecular switch, sorting misfolded proteins between the degradation and chaperone pathways during a stressed state. We hypothesised that CHIP modulation in human cancer cells may affect their sensitivity to HSP90 inhibition.

A range of levels of constitutive CHIP expression were seen across a panel of human cancer cell lines, as measured by western blotting. Additionally, CHIP levels were unaffected by treatment with HSP90 inhibitors. Interestingly, both overexpression and RNAi knockdown of CHIP caused a significant increase in cellular sensitivity to 17-AAG. Depletion of client proteins were still observed in both overexpression and knockdown cells following treatment with 17-AAG. However, differences in the rate of depletion of some, but not all HSP90 clients, were identified.

The results demonstrate that modulating CHIP levels sensitizes cells to 17- AAG but at the same time that E3-ligase redundancy may exist as a mechanism to ensure client depletion during HSP90 inhibition.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B83.
Abstract B98: Induction of metabolic and oxidative stresses by the novel inhibitor of NAD+ biosynthesis, GMX 1778, for specific killing of human glioblastomas
Authors: Yoo Stephen S. , Cerna David , Carter Donna J. , Flaherty Siobhan M. , Li Hongyun , Takebe Naoko , Watson Mark H.
Published: June 2012

Cancer therapy strategies that selectively target tumors while sparing normal tissues would provide enormous clinical benefit to cancer patients. Central to developing these strategies is identification of tumor specific vulnerabilities and molecular targeted agents targeting these vulnerabilities. GMX1777/1778, a novel inhibitor of NAD+ biosynthesis, is a potent and specific inhibitor of the nicotinamide adnine dinucleotide (NAD+) biosynthesis enzyme, phosphoribosyl transferase (NAMPRT). Since cancer cells have a high rate of NAD+ turnover, modulation of NAD+ biosynthesis would be an attractive target for GMX 1777/1778. In addition to the depletion of NAD+ level, we present evidence of inducing other metabolic and oxidative stresses in a tumor specific manner with minimal damage to normal tissues in vitro and in vivo. Taken together, the cancer therapeutic strategy presented here would provide a novel way of specific killing of tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B98.
Abstract C109: NOV-002 suppresses tumor cell growth by modulating redox-sensitive cell signaling
Authors: Townsend Danyelle M. , Bowers Robert , Pazoles Christopher J. , Tew Kenneth D.
Published: June 2012

Regulation of cell proliferation/survival is complex, impacted by multiple endogenous and exogenous stimuli capable of triggering overlapping and competing signaling pathways whose net effects can be quite cell dependent. Changes in redox status is one regulator of cell life/death decisions under both physiologic and pathologic conditions. Here we report on the anti-proliferative effects of a pharmacologically generated oxidative signal in human tumor cells using NOV-002, a glutathione disulfide-mimetic in advanced clinical trials for oncological indications. NOV-002 has demonstrated efficacy (increased survival and/or decreased tumor growth) in non-small cell lung, breast and ovarian cancers when combined with standard chemotherapeutic agents. In addition, treatment with NOV-002 mitigates chemotherapy-associated hematological toxicity. Pre-clinical data has linked this effect to proliferation of bone marrow progenitor cells subsequent to generation of an oxidative signal, intracellularly and at the cell surface of myeloid lineage cells, leading to activation of multiple kinases known to regulate cell proliferation (e.g. MAP kinases, JAK/STAT kinases). The data presented here extend redox modulation studies with NOV-002 to tumor cells.

SKOV3 cells (a human ovarian tumor cell line) were exposed to NOV-002 (250 uM). Within 5 min after a single treatment, cellular levels of reactive oxygen species (ROS) were significantly elevated indicating the generation of an oxidative signal by NOV-002. This was not, however, sufficient to influence redox-sensitive cell signaling or tumor cell proliferation rate. In contrast, more prolonged exposure to NOV-002 (daily treatment for 5 days) resulted in a sustained elevation in ROS and dose-dependent activation of the proliferation-regulating MAP kinase, JNK, as evidenced by an increase in its active, phosphorylated form. Most importantly, this redox-activated cell signaling effect was associated with a significantly decreased proliferation rate of the SKOV3 cells that persisted even after stopping treatment with NOV-002. Thus, oxidative signal generation and MAP kinase pathway activation by NOV-002 appears to result in cell-type dependent effects on proliferation/survival. In myeloid lineage/bone marrow cells, chronic exposure to NOV-002 leads to increased proliferation while in tumor cells the result is a decrease in proliferation. These dichotomous effects may contribute to the unique clinical profile that NOV-002 has demonstrated to date — increased anti-tumor efficacy and survival combined with enhanced recovery from chemotherapy-induced hematological toxicity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C109.
Abstract C116: The KidsCancerKinome: Validation of drug targets for high risk childhood cancers
Authors: Caron Huib N. , Westerhout Ellen M. , Molenaar Jan J. , Boer den Monique L. , Clifford Steve C. , Delattre Olivier , Geoerger Birgit , Lanvers Claudia , Pietsch Torsten , Scotlandi Katia , Sera Massimo , Shipley Janet , Vassal Gilles , Versteeg Rogier , Verschuur Arnauld
Published: June 2012

The aim of KCK is the pre-clinical selection and validation of drug targets and corresponding targeted drugs for 6 high risk childhood cancers. The KCK consortium focuses on six aggressive childhood tumors, killing ∼2000 children in Europe annually, i.e Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, medulloblastoma and ALL. The KidsCancerKinome (KCK) project is funded by the EU in the FP6 programme and consists of 9 tumor biology labs in 4 European countries.

The experimental approach encompasses the following points:target presence analyses (mRNA and protein expression of the human kinome)molecular validation of kinase tumor dependency (RNAi)kinase mutation analysisin vitro drug efficacy testingin vivo proof-of-principle of drug efficacy

The KCK consortium has generated gene expression profiles (Affy U133plus2 arrays) of >500 clinical tumor samples form those six tumor types. We have performed extensive analyses of mRNA expression of human kinases. Tissue microarrays with >800 tumor samples have been analysed for various kinases and for phosphorylation status of downstream proteins. Examples of interesting expression patterns of the human kinome will be presented. Detailed analyses for the first 5 kinases for which targeted drugs are available, i.e. PI3K, IGF1R, AURKA+B, and CDK2 will be presented. Lentiviral shRNA mediated knockdown of kinase protein expression has been used in cell line panels for each tumor type to select kinases with tumor dependency as validated drug targets.

Many novel kinase inhibitors are under development for adult oncology and KCK will test their in vitro activity against the tumor-driving kinases identified in this program. We are currently testing small molecule inhibitors for the first 5 kinases. For those kinases that have no small molecule inhibitors, a novel generation of siRNA based nucleic acid drugs (LNAs), produced by the Santaris company, will be applied and tested in vitro. Successful small molecule inhibitors and LNAs will be taken further to in in in vivo validation in established xenograft models of the six childhood tumor types. Pharmacokinetic studies of these drugs will finally prepare them for evaluation in future clinical studies in childhood cancer patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C116.
Abstract C136: An analysis of BCAR3-mediated regulation of p130Cas localization and focal adhesion formation in breast cancer cells
Authors: Borre Pierre Vanden , Near Richard , Lerner Adam
Published: June 2012

BCAR3 and p130Cas, two focal adhesion proteins that bind to one another and that were previously identified in a screen for genes that confer anti-estrogen resistance in ER+ breast cancer cells, were analyzed in breast cancer cell lines to determine the effect of BCAR3 expression on cell morphology and protein localization including the Src-mediated redistribution of p130Cas.

The effect of altered BCAR3 expression and role of BCAR3 and p130Cas association was studied in MCF7 cells stably over-expressing wildtype BCAR3 or BCAR3 containing an R743A missense mutation that prevents p130Cas association. Protein localization and cellular morphology were examined by immunofluorescence and Western analyses of cells were conducted to observe p130Cas post-translational modifications and the potential involvement of the TGFβ and β-catenin signaling pathways.

Stable over-expression of BCAR3 in MCF7 epithelial cells induces the loss of cohesive association between cells and the formation of membrane projections as well as large focal adhesions that contain abundant p130Cas. This BCAR3-mediated increase in p130Cas localization to focal adhesions is independent of the association of BCAR3 and p130Cas. Src, which is known to phosphorylate p130Cas, also induces augmented localization of p130Cas and this effect is also further enhanced by BCAR3 expression. Once again, physical interaction between BCAR3 and p130Cas is not necessary for the ability of BCAR3 to increase Src-mediated redistribution of p130Cas to focal adhesions. In spite of inducing a loss of epithelial morphology in MCF7 cells, BCAR3 over-expression did not induce SMAD2 phosphorylation or nuclear translocation of β-catenin, suggesting that neither the TGFβ or β-catenin signaling pathways are activated in response to BCAR3 over-expression in epithelial breast cancer cells. Consistent with these results, shRNA-mediated reduction of BCAR3 expression in MDA-231 mesenchymal cells ablates basal p130Cas phosphorylation, alters p130Cas and focal adhesion localization and induces an epithelial-like growth pattern with attenuated stress fiber formation and decreased tensin expression. The inhibition of stress fiber formation in MDA-231 cells prevents p130Cas phosphorylation. BCAR3 expression regulates the morphology and cytoskeleton of epithelial and mesenchymal breast cancer cells by affecting focal adhesion formation, p130Cas localization and the cytoskeletal response to Src signaling. The effect of BCAR3 appears not to involve either the TGFβ or β-catenin signaling pathways and is largely independent of BCAR3's direct physical interaction with p130Cas. Our current studies are seeking to examine the role of BCAR3's SH2 domain and serine-proline-rich region in the regulation of focal adhesion biology.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C136.
Abstract C149: Exon array analyses across the NCI-60 reveals potential regulation of TOP1 by transcription pausing at guanosine quartets in the first intron
Authors: Reinhold William C. , Mergny Jean-Louis , Liu Hongfang , Ryan Michael , Pfister Thomas D. , Kinders Robert , Parchment Ralph , Doroshow James , Weinstein John N. , Pommier Yves
Published: June 2012

Because Topoisomerase I (TOP1) is critical for the relaxation of DNA supercoils and because it is the target for anticancer activity of camptothecins, we assessed TOP1 transcript levels in the 60 cell line panel (the NCI-60) of the National Cancer Institute's anticancer drug screen. TOP1 expression levels varied over a 5.7-fold range across the NCI-60. HCT116 colon and MCF-7 breast cancer cells were the highest expressers; SK-MEL-28 melanoma and HS578T breast carcinoma cells were the lowest. TOP1 mRNA expression was highly correlated with Top1 protein levels, indicating that TOP1 transcripts could be conveniently used to monitor Top1 protein levels and activity in tissues. Assessment of the TOP1 locus by array comparative genomic hybridization across the NCI-60 showed copy numbers ranging from 1.71 to 4.13 and a statistically significant correlation with TOP1 transcript levels (p<0.01). Further analyses of TOP1 expression on an exon-specific basis revealed that exon 1 expression appeared to be generally higher, and less variable than the other exons, suggesting some form of transcriptional pausing regulation between exons one and two. Accordingly, we found the presence of multiple evolutionarilly-conserved potential G-quadruplex-forming sequences in the first TOP1 intron. Physico-chemical tests for actual quadruplex formation by several of those sequences yielded quadruplex formation for two of them and duplex formation for one. The observations reported here suggest the hypothesis that there is a conserved negative transcription regulator within intron 1 of the TOP1 gene, and the regulator is a quadruplex-prone region.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C149.
Abstract C156: Molecular targeting CK2 by siRNA or antisense delivered by tenfibgen ligand nanocapsules modulates NF-kappaB, TP53 and the malignant phenotype in head and neck cancer
Authors: Chen Zhong , Brown Matthew S. , Diallo Oumou T. , Hu Michael , Ehsanian Reza , Yang Xinping , Arun Pattatheyil , Lu Hai , Korman Vicci , Unger Cretchen , Van Waes Carter
Published: June 2012

We previously reported that protein kinase CK2 contributes to aberrant NF-kappaB activation, but the molecular mechanisms and antitumor effects of targeting CK2 have not been thoroughly investigated in head and neck squamous cell carcinoma (HNSCC). In the present study, we showed elevated CK2alpha, alpha' and beta mRNA and protein expression in a panel of HNSCC lines and tissues. siRNA specifically knocked down CK2 subunits and differentially inhibited IKKbeta mediated IkappaBalpha degradation, p65 synthesis and phosphorylation at serine 536 and 529, p65 and p50 binding, and NF-kappaB reporter activities. CK2alpha knockdown also upregulated two genes promoting apoptosis, TP53 and TAp63, which together with inhibition of NF-kappaB downregulated genes promoting growth, survival and adhesion, such as CyclinD1, BCL-XL, ITGA3, and ITGB4. Knockdown of CK2alpha' or beta also exhibited differential effects on gene expression. Consequently, CK2alpha siRNA most significantly inhibited proliferation, cell cycle, and migration, while inducing apoptosis and cisplatin sensitivity in vitro. Furthermore, delivery of antisense targeting CK2alpha and alpha' using tenfibgen (fibrinogen fragment of tenascin) coated nanocapsules significantly suppressed tumor growth in HNSCC xenograft models. Consistent with CK2 inhibition, antisense-treated tumor tissue specimens showed decreased expression of CK2alpha and alpha', NF-kappaB p65, Cyclin D1, Bcl-XL and Bcl2 proteins, decreased NF-kappaB p65 phosphorylation at serine 536 and 529, as well as increased TP53 and p63 by immunohistochemistry. Our study revealed the novel role of CK2 subunits in co-regulating NF-kappaB and TP53/p63 expression and signaling, as well as downstream gene expression, which result in anti-tumor effects in vitro and in vivo. Targeting the CK2 subunits may be an effective antitumor strategy with the ability to enhance the efficacy of cisplatin chemotherapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C156.
Abstract C157: Identification of a novel DNA topoisomerase II inhibitor by proteomic profiling
Authors: Kawatani Makoto , Takayama Hiroshi , Muroi Makoto , Kimura Shinya , Maekawa Taira , Osada Hiroyuki
Published: June 2012

Background: BNS-22 is a chemically synthesized derivative of the plant natural product GUT-70 isolated from the stem bark of Calophyllum brasiliense. BNS-22 possesses antiproliferative activity on human cancer cells, but its mechanism of action is unknown. We recently developed a proteome-based profiling system to predict molecular targets and mechanisms of action of bioactive small molecules. Here we report on the identification of a molecular target for BNS-22 by using the proteomic profiling approach.

Materials and Methods: HeLa cells were treated with BNS-22 and 19 well-characterized small molecules including signal transduction inhibitors and anticancer drugs for 18 h, and the cell lysates were subjected to 2-dimensional fluorescence differential gel electrophoresis (2D-DIGE). Then, we extracted 314 spots detected in all analysis gels, and performed cluster analysis based on the expression levels of those spots.

Results: Proteomic profiling analysis indicated that BNS-22 was classified into the same cluster as ICRF-193, a DNA topoisomerase II (TOP2) catalytic inhibitor. This profiling analysis also distinguished clearly the action between ICRF-193 and VP-16 (etoposide), a TOP2 poison. These results suggested that BNS-22 acted as a TOP2 catalytic inhibitor and not a TOP2 poison in cells. BNS-22 inhibited the DNA decatenation activity of human TOP2 in vitro, with an IC50 value of 2.4 M. BNS-22, like ICRF-193, induced the abnormal mitotic figure characterized by the unusually-distorted spindle and the failure of chromosome alignment and segregation, as assessed by immunofluorescence staining with anti- -tubulin antibody, anti- -tubulin antibody, and DAPI. Flow cytometric analysis showed that BNS-22-treated cells were accumulated at G2/M phase after 24 h in a dose-dependent manner. In the time course experiment, peaks corresponding to 8C were observed after 36 h in cells treated with 10 M BNS-22, indicating that BNS-22 as well as ICRF-193 induced polyploidy. BNS-22, unlike VP-16, did not cause DNA double-strand breaks, as measured by the phosphorylation of histone H2AX on Ser 139 (-H2AX), and furthermore antagonized VP-16-mediated DNA damage.

Conclusion: These results strongly suggest that BNS-22 targets TOP2 and acts as its catalytic inhibitor in cells.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C157.
Abstract C163: Gene expression signature-based Connectivity Map analysis revealed vorinostat as a novel therapeutic target agent in human gastric cancer
Authors: Claerhout Sofie A. , Lim Jae Yun , Choi Woonyoung , Kim Sang-Bae , Park Yun-Yong , Kim KyoungHyun , Mills Gordon B. , Hong Waun Ki , Lee Ju-Seog , Cho Jae Yong
Published: June 2012

Gene-expression profiling has been applied in specific settings such as to elucidate the mechanisms of biological pathways, to classify subtypes of a disease, and to predict cancer prognosis. Furthermore, the Connectivity Map using gene-expression signature can categorize the biological responses to a large number of diverse small molecule therapeutics.

To find new agents targeting gastric cancer, we analyzed global human gastric cancer gene expression profile using the Connectivity Map. From 3360 gastric cancer specific genes, it was revealed that the candidate compounds for targeting gastric cancer were histone deacetylase inhibitors (HDACi) such as vorinostat. We validated the therapeutic efficacy of vorinostat in gastric cancer cell lines. Vorinostat induced both apoptosis and autophagy in gastric cancer cell lines, but showed a different pattern in each cell line. Pharmacological or genetic inhibition of autophagy respectively by chloroquine and a siRNA against Beclin 1 increased the therapeutic efficacy of vorinostat, indicating a mechanism of drug resistance. Further transcriptome analysis will make it possible to find a predictive biomarker gene signature for vorinostat and as such can help in the future to better understand the action mechanisms of vorinostat. In conclusion, we showed that systems analysis using Connectivity Map with a human gastric cancer gene signature can be novel approach to find targeting agents for gastric cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C163.
Abstract C178: Enhanced suppression of tumor growth by concomitant treatment of human lung cancer cells with SAHA and arsenic trioxide
Authors: Chien Chia-Wen , Yao Ju-Hsin , Lee Te-Chang
Published: June 2012

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well-documented. ATO may cause DNA damage through the generation of reactive oxygen intermediates. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), modulates gene or protein expression via histone dependent or independent pathway that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. The objective of this study is to investigate whether ATO and SAHA could synergistically enhanced cell killing to cancer cells. The present studies showed that combined treatment with ATO and SAHA results in synergistic cytotoxicity in non-small cell lung carcinoma H1299 cells. By analyzing the annexin V positive cells, apoptotic cell death is significantly enhanced in combined treatment. By aid of Comet assay, we demonstrated that ATO induces DNA breaks in H1299 cells, but not SAHA. However, co-treatment with SAHA and ATO significantly increases ATO-induced DNA damage. At the doses used, ATO does not interfere with the cell cycle progression, whereas SAHA induces the expression of p21WAF1/CIP1 and leads to G1 arrest. Our present results suggest that SAHA may result in G1 arrest, which becomes more susceptible to ATO for causing DNA damage.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C178.
Abstract C186: A glycoengineered anti-CD19 antibody (MEDI-551) is effective as monotherapy or in combination treatment in mouse models of B-cell malignancies
Authors: Ward Beth K. , Mittereder N. , Kuta E. , Sims G. , Damschroeder M. , Dall'Acqua W. , Bowen M. A. , Wu H. , Tedder T. F. , Kiener P. A. , Jallal B. , Coyle T. , Herbst R.
Published: June 2012

Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen that is expressed by early pre-B cells and throughout B cell differentiation until it is down-regulated during terminal differentiation of plasma cells. Expression of CD19 is maintained following malignant transformation and therefore CD19 is expressed on the majority of B cell-derived leukemias and lymphomas.

MEDI-551 is a humanized immunoglobulin G1 kappa (IgG1) monoclonal antibody (MAb) directed against CD19. The Fc portion of MEDI-551 does not contain a fucose sugar moiety due to its expression in BioWa's Potelligent® cells. This afucosylated form of MEDI-551 exhibits enhanced CD16 binding and antibody-dependent cellular cytotoxicity (ADCC). Rituximab, another B-cell targeted therapy, is directed against CD20 and is widely used for the treatment of B cell malignancies. However, a subset of patients is unresponsive to rituximab therapy and the majority of patients will relapse following treatment. We have previously shown that the glycoengineered, ADCC-enhanced anti-CD19 MAb is effective in in vitro and in vivo models of B cell leukemia and lymphoma. The purpose of the present study was to explore the anti-tumor activity of the anti-CD19 MAb in SCID mouse models of human lymphoma as single agent and in combination with rituximab. In several models the combination of anti-CD19 with anti-CD20 resulted in prolonged suppression of tumor growth with no untoward adverse events observed. The results suggest that combination therapy targeting both CD19 and CD20 could be an effective approach in the clinic for the treatment of B cell lymphomas.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C186.
Abstract C21: Evaluation of an 8-plex cytokine immunoassay on the AVANTRA®, a novel automated biomarker workstation
Authors: Maimonis Peter
Published: June 2012

Decision Biomarkers, Inc. (DBI) has developed a novel, automated, immunoassay platform to simultaneously evaluate multiple biomarkers in a single reaction vessel. Biomarker panels are used for many research and clinical indications. We have generated data from a multiplex assay measuring a panel of 8 cytokines: IL-1β, IL-2, IL-5, IL-6, Il-8, IL-10, IL-12, and TNFα. The heart of the system is a microarray of the biomarker specific monoclonal antibodies spotted onto a glass slide which has been coated with an ultra-thin, two-dimensional film of nitrocellulose. This microarray is assembled in a plastic biochip (MAX BIOCHIP™): a self-contained device with a series of micro fluidic channels, valves, sensors, and reagent chambers containing dried, biotinylated detection antibodies, streptavidin-conjugated Cy3, and wash buffer. The biochip is processed in the AVANTRA® biomarker workstation following injection of 200 µl of sample (100 µl of plasma diluted in 100 µl of PBS). The instrument initiates, in sequence, the flow of sample, detection antibody, label, and buffer, across the array. The end result is in a fluorescent labeled complex that is then imaged by an internal CCD camera. Spot intensity is correlated with analyte concentration by instrument software. “Master” curves” electronically stored in the instrument are used to calculate analyte concentrations through a 2-point calibration scheme. Each cytokine assay has a 4-log dynamic range and individual analytical sensitivities ranging between 2.7 and 20 pg/ml. Both the intra- and interassay precision results yielded mean CV's consistently <10%

Reproducibility was demonstrated by overlapping curves following replicate standard evaluation. The unique surface chemistry, marker specific reagents, and enhanced biochip micro fluidics and instrument automation, all combine to produce a robust and easy to use multiplex immunoassay. Currently in development are a 10-plex angiogenesis panel and a 12-plex cardiac panel that will soon be available. Additionally, assays for numerous other biomarker panels are slated for future development. Custom assay development is also available.

In summary, DBI has developed a sensitive and precise, multiplex immunoassay for 8 cytokines that can run on its new, automated platform, the AVANTRA® biomarker workstation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C20.