bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C51: Molecular markers, prognosis, and efficacy of adjuvant anthracyclines in breast cancer.
Authors: Ali Alaa M. G. , Provenzano Elena , Abraham Jean , Driver Kristy , Bartlett John M.S. , Munro Alison , Poole Christopher John , Hiller Louise , Dunn Janet , Twelves Christopher , Earl Helena Margaret , Caldas Carlos , Pharoah Paul
Published: June 2012

Background: Molecular tumor markers can be used to classify breast cancer into sub-types that have distinct survival patterns. It is not known whether these sub-types respond differently to adjuvant chemotherapy. The purpose of this study was i) to evaluate the prognostic significance of breast cancer sub-types in a cohort of women taking part in the NEAT and BR9601 clinical trials comparing cyclophosphamide, methotrexate, and fluorouracil (CMF) with ECMF (epirubicin and CMF) and ii) to evaluate whether the sub-types were predictive of the added benefit of epirubicin in these trials.

Methods: We classified the tumors into six intrinsic sub-types using data generated from tumor tissue microarrays that were stained and scored for ER, PR, HER2, EGFR and CK5/6. We used Cox regression to compare relapse-free survival (RFS), breast cancer specific survival (BSS) and overall survival (OS) in the different subgroups. We also compared the effect of ECMF with CMF by subgroup.

Results: IHC data were available for 1725 cases of whom 805 were luminal 1-basal negative, 153 were luminal 1-basal positive, 174 were luminal 2, 192 were HER2-like, 230 were core basal phenotype and 171 5-negative phenotype. Median follow-up time was 7 years. The prognostic effects of the sub-types were similar to those reported for unselected breast cancer cases irrespective of adjuvant therapy. In particular, the luminal 1-basal negative tumors were associated with the best prognosis during the five years after surgery and the HER2-like tumors were associated with the poorest prognosis. ECMF has previously shown to be associated with a 33 percent relative risk reduction for OS compared to CMF. There was little evidence for significant heterogeneity of effect by tumor subtype for any end point (OS P=0.40, BSS P=0.53 RFS P=0.50). However, there was an observed trend towards the largest additional benefit from epirubicin being in women with tumors of the 5-negative phenotype (OS HR=0.39 95% CI 0.21–0.73) and the smallest being in luminal 1-basal negative tumors (OS HR=0.86 95% CI 0.64–1.16).

Conclusion: In a clinical trial in which all patients received chemotherapy, we confirmed that breast cancer sub-types show distinct behaviour with differences in short and long term survival. These differences seem to be independent of the type of chemotherapy - the benefit of ECMF over CMF was statistically similar in all disease sub-types.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C51.
Abstract C52: Enhancing the activities of endogenous antibodies.
Authors: Alley Stephen , Benjamin Dennis , Emmerton Kim , Zhang Xinqun , Law Che-Leung , Okeley Nicole , Senter Peter
Published: June 2012

Many monoclonal antibodies (mAbs) exhibit activity through antibody dependent cellular cytotoxicity (ADCC), due to the binding interactions between the Fc portion of the mAb and Fc RIIIa on natural killer cells, macrophages and neutrophils. The strength of this interaction is affected by the glycosylation pattern at Asn-297 on mAb heavy chains. Defucosylated mAbs and mAbs with bisecting N-acetylglucosamine carbohydrates bind to Fc RIIIa with higher affinities, have more pronounced ADCC activities, and have demonstrated promising in vitro and in vivo activities. We have described a novel method for generating such mAbs involving the addition of simple fucose derivatives to the culture media of antibody-producing cell lines. This leads to the production of defucosylated mAbs with significantly (20–50 fold) increased ADCC activities, while minimally impacting production and cell viability. Generation of sugar engineered antibodies in this manner is distinguished from other technologies in that there is no need for genetic engineering and cell line modification. In addition, the technology has unique in vivo applications for endogenous production of defucosylated antibodies with high affinity binding to mouse Fc receptors. Here, we show for the first time that immunocompetent mice generate defucosylated antibodies when orally administered fucose derivatives such as fluorofucose. There were no apparent toxicities associated with treatment with fluorofucose at doses that led to greater than 90% defucosylation of circulating antibodies, Applications for these novel findings will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C52.
Abstract C64: Antitumor activity of GBR 500, a monoclonal VLA-2 antibody.
Authors: Hou Sam , Mancini Laura , Pesenti Enrico A. , Mottl Harald
Published: June 2012

VLA-2 is the alpha2beta1 integrin which mediates interactions of cells with collagens, laminin and other extracellular matrix components. In tumor cells, VLA-2 has been described to be involved in neovascularization, tumor-stroma interactions and metastasis.

The objective of this study was to assess the potential of VLA-2 as a target in a range of tumor models.

We used GBR 500, a monoclonal IgG4 antibody directed against the alpha2 subunit of the alpha2beta1 integrin heterodimer. The antibody binds to human but not to mouse VLA-2. GBR 500 is currently undergoing clinical phase I studies.

We first characterized the expression of VLA-2 in a set of 96 commonly used human cancer cell-lines from different tissues/organs (including colorectal, skin, breast, prostate, pancreas, lung, cervix, kidney, ovary, CNS, bone, liver, thyroid, and blood) and screened for growth inhibition in solid tumor subcutaneous xenografts.

In an orthotopic mouse xenograft model using a DU145-luc cell line we explored the importance of VLA-2 for tumor growth in a physiological setting. We finally assessed the effect of VLA-2 antagonism on metastasis in a PC-3 luc metastasis model.

Results: VLA-2 inhibition leads to modest growth reduction in subcutaneous xenografts. In the orthotopic xenograft stronger tumor growth inhibition was achived. The strongest tumor growth inhibition effects were achieved in the mestastasis model.

Conclusion: According to our finding growth inhibition of tumors by VLA-2 inhibition increases with increased stroma exposition

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C64.
Abstract C66: Role of mTORC1 and mTORC2 in multiple myeloma and their targeting with CC214.
Authors: Pandiella Atanasio , Chen Xi , Ocio Enrique M. , Diaz-Rodriguez Elena , Lopez-Girona Antonia , Chopra Rajesh , Miguel Jesus San
Published: June 2012

The Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase which regulates cell growth, proliferation and metabolism and plays those roles by forming two functionally distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Deregulation of mTOR pathway is common in different cancers including multiple myeloma (MM). Moreover, recent studies on the role of Deptor and Grb10 in myeloma cells emphasize the importance of mTOR pathway in MM. However, a clear identification of the role of mTORC1 and mTORC2 in myeloma cells has not been achieved due to the lack of inhibitors specifically targeting these two branches. In this study, we show that both mTORC1 and mTORC2 are constitutively active in a panel of MM cell lines. Moreover, we analyzed the basal status of both branches in a cohort of MM patients (n=40) by means of multiple parameter flow cytometry using phospho-S6 and phospho-Akt as readouts for activation of mTORC1 and mTORC2, respectively. To explore the contribution of mTORC1 or mTORC2 route to the proliferation of MM cells, we employed the RNA interference of Raptor and Rictor through infection of OPM2 and MM1S with shRNA expressing lentivirus, and found that downregulation of Raptor substantially decreased cell proliferation to a higher extent than Rictor and knockdown of mTOR fully prevented cell proliferation. These data suggested a major role of mTOR pathway in the regulation of myeloma cell proliferation and support the idea of mTOR as a target for anti-myeloma therapy. For this reason, we evaluated the novel mTOR inhibitor CC214. This drug inhibited cell growth in all the cell lines tested, induced cell cycle arrest at G0/G1 by altering cell cycle related proteins, and provoked apoptosis evident by the cleavage of caspases and PARP. Besides, CC214 induced cell death in plasma cells in ex vivo culture of freshly isolated cells from bone marrow of MM patients. These results suggest that mTOR inhibitors may be of help in the future treatment of myeloma patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C66.
Abstract C83: SiRNA screen identifies a G2-M signaling node as a novel therapeutic target in medulloblastoma.
Authors: Vibhakar Rajeev , Harris Peter , Venkatraman Sujatha , Alimova Irina , Foreman Nicholas
Published: June 2012

Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although cisplatin chemotherapy and radiation have been the cornerstones of medulloblastoma intervention for over 20 years, the outcomes of these highly cytotoxic treatments are far from optimal, and there is increasing evidence that they cause long-term problems such as neurocognitive deficits and secondary tumors, even in patients with a good initial response. Thus, there is a critical need for more effective therapies to combat this disease.

To approach this problem we investigated protein kinase function in medulloblastoma using RNAi combined with genomic methods. First, we performed a kinome-wide siRNA screen and identified a cohort of genes that mediates medulloblastoma cell viability. These include a signaling node of kinases that are key components of the G2-M transition including, Aurora Kinase A, Aurora Kinase B, Plk1, Wee1, Nek2 and Mps1. Clearly the G2-M transition is critical for medulloblastoma cells, so kinases required for the process are potentially important therapeutic targets. We subsequently validated PLK1 and WEE 1 as bona fide therapeutic targets. Small molecule inhibitors of WEE 1 and PLK1 potently inhibited medulloblastoma cell growth and sensitized cells to radiation.

In conclusion, using siRNA screens we identified novel therapeutic targets for medulloblastoma therapy

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C83.
Abstract C86: DC120, a novel and potent inhibitor of AKT kinase, induces tumor cells apoptosis and suppresses tumor growth.
Authors: Deng Rong , Chang Shao-hua , Tang Jun , Feng Gong-Kan , Yang Fen , Qin Juan , Ding Ke , Zhu Xiao-Feng
Published: June 2012

PKB/AKT kinase is the core component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway which is one of the most frequently hyperactivated signaling pathways in human cancers. We designed and synthesized a series of 2-pyrimidyl-5-amidothiazole coumpounds based on the ATP binding site of AKT kinase, and the most potent compound DC120 was identified to inhibit AKT kinase activity in vitro with EC50 of 153nM by a FRET-based Z'-Lyte assay. Then the growth inhibition of DC120 on human CNE2 and MDA-MB-453 cell lines was tested in vitro. The results showed DC120 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing sub-G1 cells, characteristic morphologic changes of apoptosis in the nucleus, and cleaved caspase-3 and PARP. Further study showed that MDA-MB-453 cells transfected with constitutively activated AKT1 were more sensitive to DC120, while CNE2 cells with knock-down of AKT1 expression by shRNA were more resistant to DC120. More importantly, DC120 attenuated the phosphorylation levels of FKHR, FKHRL1, GSK-3 and mTOR in a dose-dependent and time-dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR in cancer cells. Additionally, the in vivo experiments showed DC120 at 20 mg/kg/qd remarkably inhibited the CNE2 xenograft tumor growth with the T/C value of 36.1%, accompanied by increasing TUNEL-positve cells in tumor sample. These data provide validation for the development of DC120 to treat cancers displaying elevated levels of AKT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C86.
Abstract C88: A novel multiplex RNA in situ hybridization (ISH)-based method for circulating tumor cell detection with demonstrated clinical utility for metastatic breast cancer.
Authors: Payne Rachel E. , Wang Fay , Su Nan , Krell J. , Zebrowski A. , Yague Ernesto , Ma Xiao-Jun , Luo Yuling , Coombes R. Charles
Published: June 2012

Purpose: Sensitive and specific detection of circulating tumor cells (CTCs) in a blood sample is required for realizing the full potential of CTCs as prognostic and predictive factors. Current approaches of CTC detection are largely ineffective, and are dependent on the presence of differential epithelial protein markers or upon crude morphological characteristics like cell size differences between CTCs and blood cells. These combined approaches do not detect all relevant CTCs due to their significant heterogeneity. The objectives of this study are to develop and characterize a novel RNA in situ hybridization (ISH)-based CTC detection system called CTCscope, and to demonstrate its potential clinical utility in metastatic breast cancer patients.

Experimental Design: Cultured cells of different cancer cell lines were spiked into whole blood, which was used as a model system for CTCscope development. A novel ultrasensitive fluorescent RNA-ISH method was used to detect mRNA transcripts of a multitude of tumor markers at single-cell resolution. To demonstrate feasibility of CTC detection in patient blood, duplicate blood samples were collected from 45 metastatic breast cancer patients with informed consent for CTC analysis by the newly developed CTCscope system and the CellSearch system. Association of CTCs with tumor marker CA15–3 and progression-free survival (PFS) was assessed.

Results: CTCscope detected mRNA transcripts of 8 epithelial tumor markers and 3 epithelial-mesenchymal-transition (EMT) markers, and resulted in increased sensitivity for CTC detection. CTCscope was used to detect CTCs without enrichment, and did not result in false positives from detection of apoptotic or dead cells. In patient blood samples, CTCs detected by CellSearch but not CTCscope were positively correlated with CA15–3 levels. CTCs detected by either CTCscope or CellSearch predicted PFS, but only CTCs detected by CTCscope remained significantly associated with PFS (hazard ratio 2.01, 95% CI 1.14–3.92, p=0.038) after adjusting for CA15–3 levels.

Conclusions: The CTCscope assay may offer unique advantages over existing CTC detection approaches. By enumerating and characterizing only viable CTCs, CTCscope may provide additional prognostic and predictive information.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C88.
Abstract C93: The design and synthesis of a novel fluorogenic probe targeting inactive forms of Burton's tyrosine kinase for high-throughput binding assay.
Authors: Kawahata Wataru , Asami Tokiko , Sawa Masaaki
Published: June 2012

The aberrant activation of B-cell receptor (BCR) signaling is thought to promote cancer cell expansion and survival in B cell lymphoma and chronic lymphocytic leukemia. The Burton's tyrosine kinase (Btk) is a member of the Tec family of cytoplasmic tyrosine kinase and plays a crucial role in the BCR signaling which is essential for B-cell development and activation. Therefore inhibitors of Btk are considered as potential therapeutic agents for the treatment of B cell lymphoma, chronic lymphocytic leukemia and autoimmune diseases. Targeting inactive conformations of kinase is an attractive approach for drug discovery to develop a selective kinase inhibitor, because it is believed that structures of inactive conformation are much diverse than that of the active kinase form. To identify compounds that preferentially bind to the inactive conformation of Btk, we have designed and synthesized a novel fluorogenic probe for high throughput screening.

Methods: CGI1746 has been reported to bind to the inactive form of Btk, so we chose this compound to probe the inactive conformer of Btk. To detect its binding to the active site of Btk, we covalently attached an environmentally-sensitive fluorophore, nitrobenzoxadiazole (NBD) to CGI1746 via various linkers. The fluorescence of NBD is extremely sensitive to the local environment around the chromophore. Namely, NDB is almost nonfluorescent in aqueous solution, but become highly fluorescent in nonpolar solvents or when bound to proteins. Therefore we expected that this unique environment-sensitive fluorescent property would make the experiment process simple, easy handling, and of course, does not require radioisotope labeling.

Results: We synthesized NBD-labeled CGI1746 derivatives with different likers, and tested their inhibitory activity against Btk. Most of the compounds syntheszed were found to be execellent inhibitors of Btk with an IC50 value in the low nanomolar concentration range. As expected, the NBD-labeled CGI1746 probe showed strong fluorescence when it mixied with Btk protein but quenched by adding of free CGI1746. Interestingly, the fluorescence intensity of the probes varied considerably along with the linker structures. To validate if the probes can detect compounds that bind preferenctially to inactive form of Btk, we screened the small compound library using activated Btk and unactivated Btk. Detailed data of screening will be presented in this poster.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C93.
Abstract C97: Studies on the signaling pathway for cancer cell migration based on chemical genomic approach.
Authors: Magi Shigeyuki , Tashiro Etsu , Imoto Masaya
Published: June 2012

Cell migration is known to be related to not only physiological phenomena such as embryonic development, immune reaction, and wound healing, but also pathological phenomena such as asthma, vascular disease, and cancer metastasis. However, because genetic mutations of each cancer cell causing high migration ability depend on cell types, it still remains unclear that which pathways are the unity of cancer cell migration signaling irrespective of cancer cell types, and which pathways are the diversity depend on cell types. The aim of this study is to reveal the diversity and unity of regulatory signaling for cancer cell migration based on chemical genomic approach.

To understand the diversity and unity of regulatory signaling for cancer cell migration, the effects of 38 small compounds, whose target protein are already identified, on cell migration ability of 10 types of cancer cells were assessed quantitatively by wound healing assay. Two-way hierarchical clustering was done on migration ability profile by small compounds or cancer cell types. As a result, cancer cells tested in this study were classified into three clusters (group A∼C). Interestingly, epidermoid carcinoma A431 cells and esophageal squamous carcinoma EC109 cells (group B) were classified different cluster from esophageal squamous carcinoma TE8 cells and thyroid medullary carcinoma T.T cells (group C), whereas all four cell lines were induced migration by EGF stimulation. On the other hand, compounds were clustered into five clusters (cluster I∼V). The compounds classified into cluster I (e.g., V-ATPase inhibitor and bisphosphonates) showed little inhibitory effect on all types of cell migration. The compounds clustered into cluster II (e.g., EGFR inhibitor and ROCK inhibitor) showed stronger inhibitory effect on the migration of group B and C than A. The compounds classified into cluster III (e.g., JNK inhibitor) showed strong inhibitory effect on all types of cell migration tested in this study. The compounds clustered into cluster IV (e.g., PI3K inhibitors and mTOR inhibitor) showed stronger inhibitory effect on the migration of group B than C. On the contrary, the compounds classified into cluster V (e.g., MEK inhibitor and p38 inhibitors) showed stronger inhibitory effect on group C than B. These results indicate that JNK play the common role in cancer cell migration, and belonging to cluster II, IV, and V would have different function in each type of cancer cell migration. We are now investigating how these molecules actually operate in the signaling pathway in each cancer cell, and trying network analysis by the combination of chemical biology and systems biology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C97.
Abstract CN05-04: Opportunities for targeting homologous recombination in human cancers.
Authors: Powell Simon N.
Published: June 2012

BRCA2 mediates homologous recombination (HR) via its direct interaction with Rad51. There is ample genetic and biological evidence that BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the functional connection between the two proteins is poorly understood. Interestingly, a mutation of BRCA1 (S988A) localized normally to ionizing radiation induced nuclear foci (IRIF), but the BRCA2 protein could not be recruited to BRCA1 at the damage site. Phosphorylation of BRCA1 at the S988 site is carried out by Chk2, which was also required for BRCA2 to localize to sites of DNA damage and support homologous recombination. These observations suggest that CHK2-BRCA1-BRCA2-Rad51 function in a single pathway of homologous recombinational repair to prevent the development of breast cancer. Loss of function of BRCA1/BRCA2 is a tumor-specific finding, making the role of alternative repair pathways in these cells critical for their survival. We show that loss of Rad52 function is synthetic lethal with BRCA1, PALB2 or BRCA2 deficiency, whereas there is no impact on cell growth in wild-type cells. The frequency of both spontaneous and double-strand break-induced homologous recombination, plus ionizing radiation induced Rad51 foci, decreased when Rad52 was depleted in BRCA2-deficient cells, with no effect in BRCA2-complemented cells. The observation that Rad52-inactivation is synthetic lethal with BRCA2 makes Rad52 a target for therapy in these tumors. The BRCA1-BRCA2 pathway can be functionally inactivated in sporadic breast cancer, perhaps as frequently as 20% of cases, suggesting that the pool of patients with defects in homologous recombinational repair is larger than initially expected. The functional assessment of defects in homologous recombination was assessed by ionizing-radiation (ex-vivo exposure) induced Rad51 foci and confirmed by characteristic patterns of array comparative genomic hybridization. The inactivation of the BRCA1-BRCA2 pathway allows the use of specific therapies designed to exploit the defects in DNA repair.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN05-04.
Abstract KN01: Overcoming barriers to new drug development.
Authors: Hait William N.
Published: June 2012

The forty years that followed the signing of the National Cancer Act witnessed substantial progress in our understanding of the biology of human cancer. This knowledge has translated into several important advances for the treatment of patients with both hematological and solid tumor malignancies. Yet, during the last 15 years we have not seen a proportional increase in the approval rate of new drugs. The drop-off in efficiency of drug development coincided, unexpectedly, with the adoption of target-based screening, which replaced a physiological or phenotypic approach. The power of molecular biology enticed drug developers to believe that the ability to express a cloned gene product to high purity and the utility of structural biological approaches to rational drug design, led to remarkably potent and selective inhibitors. Unfortunately, the understanding of how the target was wired into a specific tumor type was less obvious, making even optimal target engagement in the clinic insufficient to produce striking benefits to patients. The phenotypic approach is agnostic to target but instead relies on a readout that predicts efficacy in the clinic. For both the target-based and for the phenotypic approaches, the rate limiting step in cancer drug discovery is a predictive pre-clinical model of a specific tumor type. The challenges that remain and the advances we have made in translational research is the subject of this presentation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr KN01.
Abstract PL02-01: Changing cell fate in cancer: Lessons from melanoma.
Authors: Zon Leonard I.
Published: June 2012

Melanoma is a devastating disease with great morbidity and mortality. We are using the zebrafish as a model to study melanoma, making use of its excellent genetics, development, and large numbers of adults with cancer. A human constitutively active BRAF was fused to the melanocyte specific MITF promoter. This was injected into zebrafish embryos, generating transgenic zebrafish. These fish were crossed to a P53 mutant line generating a melanoma model. Microarray analysis of this melanoma demonstrates similarities to gene expression signatures in human melanoma. Mating the BRAF P53 fish to an MITF mutant led to complete abrogation of melanoma. The lack of melanocyte progenitors due to MITF deficiency led to an absence of tumor formation. A vector called Mini Cooper was generated in which an MITF minigene could rescue melanocyte formation in the BRAF P53 MITF mutants and a gene of interest could be probed for enhanced or expressed tumor formation. Screening an interval of human chromosome 1 (54 independent genes). That is amplified in 30% of patients) in the minicooper plasmid injection found SETDB1 a histone methyltransferase that is thought to suppress gene expression. ChIPSeq analysis in human melanoma samples demonstrated that SETDB1 binds to genes such as the Hox locus and alters their expression. SETDB1 is a potential target for anti-melanoma therapy. In another screen, small molecules were added to zebrafish embryos in an effort to abrogate neural crest development. One small molecule was found to inhibit an enzyme (DHODH), and an arthritis drug (leflunomide) that also inhibits DHODH was found to similarly block neural crest development. This DHODH inhibitor (leflunomide) worked synergistically in xenograft studies to block melanoma formation. This combination therapy will enter into the clinic as a trial for patients with metastatic melanoma. Using genetics, it was found that the DHODH inhibitor which blocks uridine production has an effect on transcription elongation and the targets of this include genes that are activated by MYC and by MITF. The zebrafish has led to the discovery of two new targets for melanoma therapy, and one of those is proceeding to the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL02-01.
Abstract PR-8: Development of a multiplex panel of putative biomarkers for assessing pharmacodynamic effects of anticancer therapies targeting apoptosis.
Authors: Srivastava Apurva K
Published: June 2012

Background: Regulators of apoptotic cell death are high interest targets for cancer therapy, and several mechanism-based investigational agents in clinical development directly target the molecular components of apoptosis pathways. We have undertaken development of a multiplex assay of candidate biomarkers of this mechanism for quantifying the commitment, onset, and induction of apoptosis by both intrinsic and extrinsic pathways. Quantitation of these pharmacodynamic biomarkers in early clinical trials will assist hypothesis testing and confirm the intended mechanism of action of investigational agents. The panel may also be adaptable to diagnostic applications to identify patients most likely to respond to a particular apoptosis inducer.

Methods: The panel being developed includes quantitative determinations of Bcl-xl, Mcl-1, Survivin, Smac dimer, cleaved Lamin-B, active/total Caspase-3, BIM, and six oligomeric forms of Bax and Bak with Bcl2 family proteins in lysates of tumor biopsies. Calibrators for oligomeric proteins, which are formed in vivo by the non-covalent association of BH3 domain proteins, are being developed as recombinant fusion proteins. The housekeeping gene GAPDH is used as an internal control. The multiplex, sandwich immunoassay is built on the Luminex® xMAP technology platform using MagPlex bead, which allows for significant automation in the detection of multiple proteins in a 96-well plate format, with increased sensitivity, smaller sample volumes, and less preparation time.

Results: Suitable, high quality capture antibodies against proteins in the panel as well as detection antibodies labeled with fluorescent dyes have been identified and tested for sandwich formation using recombinant calibrators and cell lysates. Capture and reporter antibody pairings are currently standardized for optimal assay performance. The feasibility of developing several oligomeric calibrators (among Bak-Bcl-xl, Bak-Mcl-1, Bak-Bak, Bak-Bax, Bax-Bax, and Bax-Bcl2) as recombinant fusion proteins has been established.

Conclusions: The proposed multiplex panel represents the most comprehensive selection of putative biomarkers for the apoptosis pathway, to date. Our strategy to quantify oligomeric forms of Bcl-2 family proteins in tumor lysates represents a novel approach since it evaluates a pathway based on alterations in protein dynamics, instead of the levels of various proteins. Once developed, validated, and fit to purpose, the panel will facilitate drug discovery and the real-time pre-clinical and clinical evaluation of drugs targeting apoptosis.

Funded by NCI Contract No HHSN261200800001E.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-8.
Abstract 1005: Plk3 functions as an essential component of the survival and hypoxia regulatory pathway by direct phosphorylation and regulation of PTEN and HIF-1α
Authors: Yao Yixin , Xu Dazhong , Costa Max , Dai Wei , Lu Luo
Published: June 2012

Plk3, a member of the Polo-like kinase family, plays an important role in regulating cell cycle progression and cell cycle checkpoints in response to genotoxic stresses. Plk3 is also strongly implicated in tumorigenesis because PLK3-/- mice develop tumors in multiple organs at an enhanced rate. We have previously shown that many of the tumors developed in the PLK3 null mice are large in size and are highly vascularized, suggesting that this kinase may be involved in regulating the angiogenesis pathway. By studying PLK3 null primary isogenic murine embryonic fibroblasts (MEFs), we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3-/- MEFs contained a reduced level and activity of PTEN that closely correlated with increased PDK1 and Akt1 activation as well as decreased GSK3β activity. The activated Akt1 in PLK3 null MEFs was closely associated with enhanced stability of HIF-1α under hypoxic conditions. Purified recombinant Plk3 phosphorylated PTEN and HIF-1α in vitro and phospho-mutant analysis indicated that Plk3 directly regulates these proteins in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia and angiogenesis signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1005. doi:10.1158/1538-7445.AM2011-1005
Abstract 1011: NNK promotes migration and invasion of cancer cells through activation of c-Src, PKCι and FAK loop
Authors: Shen Jie , Xu Lijun , Sun Shi-Yong , Khuri Fadlo , Deng Xingming
Published: June 2012

Cigarette smoking, either active or passive, is the most important risk factor in the development of human lung cancer. Mounting evidence indicates that cigarette smoke constituents not only contribute to tumorigenesis but also may increase the spread of cancer in the body. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen. NNK, an important component in cigarette smoke, may also promote tumor metastasis by regulating cell motility. Here we found that NNK can induce activation of a functionally interdependent protein kinase cascade, including c-Src, PKCι and FAK, in association with increased migration and invasion of human lung cancer cells. c-Src, PKCι and FAK are extensively co-localized in the cytoplasm. Treatment of cells with α7 nAChR specific inhibitor α-bungarotoxin (α-BTX) blocks NNK-stimulated activation of c-Src, PKCι and FAK and suppresses cell migration and invasion. Intriguingly, NNK enhances c-Src/PKCι and PKCι/FAK bindings, indicating a potential mechanism by which these kinases activate each other. Specific disruption of c-Src, PKCι or FAK expression by RNA interference significantly reduces NNK-induced cell migration and invasion. These findings suggest that NNK-induced migration and invasion may occur in a mechanism through activation of a c-Src/PKCι/ FAK loop, which can contribute to metastasis and/or development of human lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1011. doi:10.1158/1538-7445.AM2011-1011
Abstract 1015: P38 mitogen-activated protein kinase signaling and viability of lung cancer cells from never or light smoker patients
Authors: Planchard David , Soria Jean-Charles , Dorvault Nicolas , Camara-Clayette Valérie , Fouret Pierre
Published: June 2012

Purpose: Expression levels of DNA repair proteins and activated mitogen-activated protein kinases (MAPKs) may differ according to smoking status. We investigated whether P38 MAPK activity contributed to the viability and sensitivity to cisplatin of lung cancer cells lines from never or light smokers and to ERCC1 mRNA expression.

Methods: Activated P38 MAPK was tested by means of immunohistochemistry as a predictor of ERCC1 levels in 117 lung adenocarcinomas. Cell viability of NCI-H1975, NCI-H1793, NCI-H1650 and NCI-H1651 was measured after treatment with the P38 MAPK inhibitor SB202190 and cisplatin. The role of P38α (MAPK14), P38β (MAPK11) isoforms and ERCC1 was evaluated using ARN interference. ERCC1 mRNA was measured by quantitative PCR.

Results: ERCC1 protein expression was predicted by activated P38 MAPK in lung adenocarcinoma tissues. The P38 specific inhibitor SB202190 induced a strong decrease (P<0.01) in cell viability of NCI-H1975 (43%) and NCI-H1650 (63%) compared to control. When SB202190 was combined with cisplatin, cell viability decreased (P<0.01) in every cell line including cisplatin resistant NCI-H1793. The combination of drugs reduced cell viability more than cisplatin (P<0.01) for NCI-H1793, NCI-H1650 and NCI-H1651, and it was synergistic for NCI-H1793 and NCI-H1651. Genetic inhibition targeting both MAPK11 and MAPK14 reduced the viability of every cell line (P<0.01) with a dose-response for three of them. The decrease in cell viability with genetic inhibition at 25 nM was higher (P<0.01) than with cisplatin for NCI-H1793 (67% vs. 35%) and NCI-H1650 (55% vs. 38%). In separate experiments, MAPK11 downregulation reduced viability of every cell line (P<0.01), while MAPK14 downregulation reduced viability of NCI-H1651. The effect of cisplatin was higher after MAPK11 downregulation for NCI-H1651 and after MAPK14 downregulation for NCI-H1650. In addition, both SB202190 and MAPK11 inhibition reduced excision repair cross-complementing 1 mRNA levels.

Conclusion: lung cancer cells from never or light smokers rely on P38 MAPK signaling for their survival whether or not they harbor EGFR mutations. MAPK11 is involved in that pathway and might contribute to ERCC1 expression. Sensitization to cisplatin can be achieved by pharmacological inhibition of P38 MAPK signaling in some lung cancer models from never smokers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1015. doi:10.1158/1538-7445.AM2011-1015
Abstract 1016: Bortezomib (Velcade) induces p27Kip1 expression through S-phase kinase protein 2 degradation in papillary thyroid cancer
Authors: Hussain Azhar , Bu Rong , Bavi Prashant , Ahmed Maqbool , Al-Obaisi Khadija A.S. , Al-Sobhi Saif , Al-Nuaim Abdulrahman , Ahmed Mohammed , Amin Tarek , Uddin Shahab , Al-Kuraya Khwala S.
Published: June 2012

S-phase kinase protein 2 (SKP2), an F-box protein, targets cell cycle regulators including cycle-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancers. In this study, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in Papillary Thyroid Cancer (PTC) in a panel of PTC cell lines, a large cohort PTC patient samples and NUDE mouse model using tissue microarray, MTT, flow cytometry and DNA fragmentation assays and western blotting. Our data showed that bortezomib caused a dose-dependent growth inhibition in all cell lines studied. The IC 50 was 83.7nM and 61.4nM for B-CPAP1 and TPC-1 respectively. Cell cycle assay showed that the sub-G1 population of B-CPAP1 cell increased from 4.09% in untreated control sample to 36.74% after 25nM treatment for 2 days and 71.91% after 50nM treatment for 2 days. Further confirmation using FITC-conjugated Annexin V and PI assay showed that apoptotic cell increased form 8.43% in untreated cells to 47.6% and 64.82% after 25nM and 50nM treatment respectively in B-CPAP1 cells and from 12.72% in control to 48.16% and 64.02% in TPC-1 cells, indicating a dose-dependent apoptosis induced in both cell lines. Treatment of PTC cell line cells with bortezomib or expression of small interfering RNA of SKP2 caused down-regulation of SKP2 and accumulation of p27Kip1. Furthermore, treatment of PTC cells with bortezomib caused apoptosis by involving the mitochondrial pathway and activation of caspases-9 and 3, and cleavage PARP. Pretreatment with zVAD-fmk abrogated bortezomib induced apoptosis. In addition, treatment of PTC cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Currently the animal study and data analysis of patient samples are still ongoing. Our preliminary results suggest that SKP2 and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention for treatment of TPC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1016. doi:10.1158/1538-7445.AM2011-1016
Abstract 1018: Regulation of STAT3 by PPARdelta in human hepatocellular carcinoma
Authors: Han Chang , Huang Shuo , Wu Tong
Published: June 2012

Peroxisome proliferator-activated receptor delta (PPARdelta) is a nuclear transcription factor that is recently implicated in tumorigenesis. Previous studies from our lab have shown that PPARdelta activation increases the growth of human hepatocellular carcinoma (HCC) cell lines (Hep 3B, Huh7 and HepG2). The current study was designed to investigate the relationship between PPARdelta activation and STAT3 signaling pathway in human HCC cells. Our data showed that PPARdelta overexpression or activation by its pharmacological ligand (GW501516) significantly increased STAT3-mediated transcriptional activity in Hep3B cells, as determined by luciferease reporter activity assay; this effect is dose-dependent (p<0.01). In contrast, depletion of PPARdelta by its siRNA in Hep3B cells significantly reduced GW501516-induced STAT3 transcriptional activity. PPARdelta overexpression or GW501516 treatment increased the expression of STAT3 protein and induced the translocation of STAT3 from cytosol to nuclei in Hep3B cells. Additionally, PPARdelta overexpression or GW501516 treatment also increased the binding of PPARdelta to STAT3-responsive element through the release of transcriptional repressor Bcl-6. Moreover, overexpression of PPARdelta upregulated the expression of several STAT3 downstream anti-apoptotic molecules including Mcl-1, Suvivin and Bcl-XL. These findings reveal a cross-talk between PPARdelta and STAT3 signaling pathway for human HCC cell growth and such an interaction may play important role in hepatocarcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1018. doi:10.1158/1538-7445.AM2011-1018
Abstract 1021: Bortezomib-mediated inhibition of NF- κB activity leads to induction of apoptosis in activated B-cell like diffuse large B-cell lymphoma
Authors: Bu Rong , Hussain Azhar R. , Ahmed Maqbool , Al-Obaisi Khadija A.S. , Uddin Shahab , Al-Kuraya Khawla S.
Published: June 2012

Diffuse large B-cell lymphoma (DLBCL) is considered to be the most common type of lymphoma in adults, accounting for 30-40% of cases of non-Hodgkin lymphoma, and classified as three distinct subtypes: germinal center B cell-like (GCB)-, activated B cell-like (ABC)-, and primary mediastinal B-cell lymphoma (PMBL). ABC DLBCL has a worse survival after upfront chemotherapy and is characterized by constitutive activation of the antiapoptotic nuclear factor-kappa B (NF-κB) pathway. Yet the implication of NF-κB inhibition in ABC DLBCL need to be further elucidated. Therefore, a panel of ABC DLBCL cell lines was used to examine the effect of bortezomib, a proteasome inhibitor which blocks degradation of phosphorylated IκBα and consequently inhibits NF-κB activity, and small interfering RNA (siRNA) on cell viability, apoptosis and downstream signaling pathway. Our data showed that bortezomib caused a dose-dependent growth inhibition in all cell lines studied. The IC 50 was 3.33 ± 0.29 nM, 2.73 ± 0.32nM, 2.60 ± 0.87nM and 4.35 ± 0.65nM for HBL-1, Riva, Sudhl-2 and OCI-LY3 respectively. Cell cycle assay showed a dose-dependent increase of sub-G1 population of all four cell lines studied after 48 hours treatment, and this increase in sub-G1 population was accompanied by a dose-dependent decrease in the percentage of cells in the G2-M phase indicating the feature of apoptosis. Further experiment using FITC-conjugated Annexin V and PI assay confirmed that apoptotic cell increased in a dose-dependent manner in all cell lines after 48 hour treatment. Moreover, the electrophoretic mobility shift assay showed that bortezomib treatment inhibited constitutive nuclear NF-κB in ABC cell lines. Furthermore, treatment of ABC cell lines with bortezomib or expression of small interfering RNA of NF-κB also down-regulated expression of NF-κB-regulated gene products, such as IκBα, Bcl-2, Bcl-Xl, XIAP and Survivin leading to apoptosis via the mitochondrial apoptotic pathway. Altogether, these results suggest that NF-κB may be a potential target for therapeutic intervention in DLBCL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1021. doi:10.1158/1538-7445.AM2011-1021