bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4361: Retinoblastoma (RB) genomic progression analysis using Tissue Microarrays (TMA) comprising 121 RB patient tumors
Authors: D'Silva Crystal N. , Yoshimoto Maisa , Natalino Renato , Soares Fernando , Antoneli Celia , Ribeiro Karina , Halliday William , Gallie Brenda L. , Squire Jeremy A.
Published: February 2011

Retinoblastoma is the second most common childhood intraocular malignancy and development of the tumor is initiated by bi-allelic loss of the RB1 gene. Bi-allelic RB1 loss may not always directly lead to malignant retinoblastoma, rather other genetic changes are likely involved. Subsequent mutational events, specifically gain and loss of chromosomal regions harboring key oncogenes and tumor suppressor genes, appear to underlie the progression of precursor lesions such as retinoma to retinoblastoma. Research thus far has implicated several cytogenetic aberrations in this sequence of molecular events, the two most recurrent and notable being the augmented copy number status of KIF14 and MDM4 on 1q and of DEK and E2F3 on 6p. A small subset of retinoblastoma also exhibits high-level amplification of the MYCN gene on chromosome 2p. KIF14, MDM4, DEK, E2F3 and MYCN have been widely characterized as oncogenes in a variety of cancers. The functional roles of the proteins encoded by these genes would also confer tumor cells manifesting these genomic changes with a selective survival and proliferative advantage. Our project is testing the hypothesis that 1q and 6p gain and 2p amplification - defined by extra copies of KIF14 and MDM4, DEK and E2F3 and MYCN respectively - are associated with clinical parameters of progression. The study comprises formalin-fixed paraffin-embedded fluorescence in situ hybridization (FFPE-FISH) analysis of pre-constructed tissue microarrays comprising 121 patient tumours, in order to determine the genomic copy number status of the five aforementioned genes. Results show 1q gain (3-10 copies) in 102/121 (84%), 6p gain in 115/121 (95%), MYCN gain in 14/121 (12%) and MYCN amplification (>10 copies) in 16/121 (14%) patient tumors. Assessment of potential correlations between the copy number status of these genes and clinical variables of disease progression include patient age, pathology at the time of diagnosis, optic nerve invasion, choroid involvement, laterality of the disease, number of tumor foci present at the time of diagnosis and histopathological parameters. To our knowledge, there has been no recent study associating genomic change of these five genes with clinical parameters of progression in a sample size of statistical significance. The large cohort and consequent statistical power of our results will facilitate a better understanding of the biological role of these genes in the progression from retinoma to retinoblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4361.
Abstract 4362: Plk1 is overexpressed in retinoblastoma and its inhibition by siRNA or small-molecule inhibitor results in growth arrest and apoptosis
Authors: Berglund Andrew , Dalgard Clifton L.
Published: February 2011

Retinoblastoma (RB) is a pediatric cancer of the retina where current chemotherapeutic regimens are broad spectrum with significant side effects. Molecular target-based approaches for RB therapy are absent and may be rational for this disease. We performed a siRNA library screen for growth inhibition of human Y79 RB cells. Knockdown of Plk1 or AURKA (but not other mitotic regulatory factors) lead to greater than 90% cell growth inhibition and prompted further investigation. We determined Plk1 expression in retinoblastoma and normal retina tissue by immunohistochemistry. We also determined the effects of siRNA-mediated Plk1 knockdown and small molecule-mediated Plk1 inhibition on Y79 and Weri-Rb1 cell growth using a tetrazolium dye-based assay. Effects on proliferation and cell cycle distribution were determined by flow cytometric analysis of EdU incorporation and DNA content. Activation of DNA damage response and apoptosis was determined by western blot analysis of phospho-H2AX, cleaved caspase-3, and PARP. We observed moderate and high Plk1 protein expression in several human retinoblastoma tumor samples as compared to minimal or low expression found in normal retinal tissue. Increased Plk1 expression was observed in RB cell lines as compared to normal retina cells. 10 nM Plk1 siRNA and 30 nM BI-2536 potently inhibited Y79 and Weri-Rb1 cell growth and significantly decreased cell viability. A decrease in S phase EdU incorporation and proliferation and an increase in G1 phase population was observed in flow cytometric analysis of Plk1 inhibited RB cells. A significant increase in phospho-H2AX, cleaved caspase-3, and cleaved PARP protein levels was observed in Y79 and Weri-Rb1 cells treated with Plk1 siRNA or BI-2536. These results suggest that Plk1 may be a rational target for RB therapy and encourages preclinical studies in animal models of RB.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4362.
Abstract 4369: MMP9, MMP14, and MMP15 genes are induced by nicotine in an E2F-dependent manner
Authors: Johnson Jackie L. , Pillai Smitha , Chellappan Srikumar
Published: February 2011

Cigarette smoking is strongly correlated with the onset of non-small cell lung cancer (NSCLC). Nicotine, the addictive component of cigarettes, has been found to induce proliferation, confer resistance to apoptosis, and induce EMT like changes in breast, pancreatic, and lung cancer cell lines. In addition, nicotine also induces morphological changes characteristic of a migratory, mesenchymal phenotype in NSCLCs. Correspondingly, the exact molecular mechanism for this acquired invasiveness in NSCLCs has been partially elucidated, but still remains somewhat unclear. To determine whether this increased invasion is due to up-regulation of proteolytic proteins, we first examined the mRNA levels of matrix metalloproteinase genes, which are crucial proteins for the mechanical degradation of the ECM, after exposure to nicotine. MMP9, MMP14, and MMP15 mRNA levels were significantly induced by treatment with nicotine when compared to serum starved A549 lung adenocarcinoma cells. Transient transfection experiments in H1650, A549, and MDA-MB-231 cell lines also show that a 2kb MMP9-luc promoter is induced by treatment with nicotine. In order to identify a possible factor responsible for this induction, we next analyzed the promoters of MMP genes. Chip assays show that these MMPs had multiple functional E2F binding sites. Wound healing assays on these cells showed that cells lacking E2F1 were impaired in their ability to migrate on plastic. In addition, cells lacking E2F1 showed a lower capacity for invasion, as determined by Boyden Chamber assays. The effect of depleting E2F1 on MMP expression was assessed by Real-time PCR assays. It was found that MMP 9, 14, and 15 were down-regulated in A549 cells lacking E2F1. Furthermore, these MMP9, 14, and 15 promoter reporters were induced upon co-transfection with either E2F1 or E2F3, both classical E2F activators. These data suggest that the Rb/E2F pathway is a likely downstream target of nicotine and mediates invasion by enhancing the levels of MMPs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4369.
Abstract 4370: Arsenite stabilizes HIF-1α protein through p85α-mediated upregulation of inducible Hsp70 protein expression
Authors: Guo Wei , Zuo Zhenghong , Yang Zhuo , Xia Qing , Liu Jinyi , Li Jingxia , Zhang Dongyun , Shi Xianglin , Huang Chuanshu
Published: February 2011

Arsenite has long been known as a human carcinogen. The hypoxia-inducible factor-1 (HIF-1) is an important transcription factor upon hypoxia and other stresses, which is proved to be associated with the pathogenesis of several cancers. Although hypoxia-inducible factor-1α (HIF-1α) has been reported to be activated by arsenite both in vivo and in vitro, the detailed molecular mechanisms leading to HIF-1α expression and activation due to arsenite exposure have not been well understood. In the present study, we found that arsenite is able to induce HIF-1α protein accumulation in both mouse epidermal Cl41 cells and mouse embryonic fibroblasts (MEFs). Knockout of p85α in MEFs (p85α−/−) led to the deficiency of HIF-1α protein accumulation induced by arsenite exposure, suggesting that p85α is crucial for arsenite-induced stabilization of HIF-1α protein. At the same time, our studies also show that p85a exhibited an inhibition of HIF-1α protein degradation via PI-3K/Akt-dependent pathway in cellular response to arsenite exposure. Our further studies revealed that arsenite exposure was able to induce inducible Hsp70 expression, and the deficiency of HIF-1α protein in p85α−/− MEF cells could be restored by introduction of inducible Hsp70 into p85α−/− MEF (p85α−/− (Hsp70)). Moreover, our results also indicated that p85α is crucial for arsenite-induced activation of heat shock transcription factor 1 (HSF-1), which is responsible for the transcription of inducible Hsp70. The transcriptional up-regulation of hsp70 by activated PI-3K/Akt pathway is strictly mediated by heat shock transcription factor-1 (HSF-1). Taken together, our studies demonstrated that p85α-mediated HIF-1α stabilization due to arsenite exposure is specific through p85α mediation of HSF-1 activation and subsequently transcriptional up-regulation of the inducible Hsp70. During this course, p85 regulated inducible Hsp70 expression transcription through PI-3K/Akt/HSF-1 pathway upon arsenite exposure.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4370.
Abstract 4372: Mechanisms involved in MMP-2 downregulation in arsenic-exposed human urothelial cells
Authors: Wang Hsiu-Hua , Lee Te-Chang
Published: February 2011

Arsenic has been reported to induce aberrant gene expression through various mechanisms including genetic, epigenetic regulation and signaling transduction pathways. Our previous studies have shown that long-term exposure of human urothelial cell line SV-HUC-1 (HUC-1 cells) to low dose arsenic resulted in altered cell morphology, reduced cell migration activity, and suppressed expression of matrix metalloproteinase 2 (MMP-2). To explore how MMP-2 is suppressed in arsenic exposed HUC-1 cells (iAs cells), we found that MMP-2 gene expression could be partly reverted by treatment with either demethylating agent 5-aza-dC or TGF-β alone. The enhanced expression of MMP-2 was additive by treatment of iAs cells with in combination of 5-aza-dC and TGF-β. Although we confirmed that MMP-2 promoter was hypermethylated by MSP assay, we unexpectedly found that Smad2 and Smad4 proteins were significantly suppressed in iAs cells, indicating that the canonical TGF-β pathway was deficient in iAs cells. The negligible expression level of Smad4 was confirmed by aid of a luciferase reporter containing Smad4-binding element. Alternatively, TGF-β-induced MMP-2 gene expression in HUC-1 and iAs cells could be suppressed by PKC inhibitor. Our current results suggested that TGF-β may mediate through PKC activation to induced MMP-2 expression. Taken together, MMP-2 expression in iAs cells could be reactivated by 5-aza-dC and TGF-β, indicating that arsenic exposure may be through epigenetic and signaling pathway alteration to regulate the expression of MMP-2.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4372.
Abstract 4373: JNK1 participates in the VHL-independent HIF-1α degradation pathway by regulating Hsp90/Hsp70 turnover and the HDAC6-dependent chaperone activity
Authors: Zhang Dongyun , Cai Tongjian , Li Jingxia , Costa Max , Huang Chuanshu
Published: February 2011

Nickel has been proved to be a human carcinogen. Hypoxia induced factor-1α (HIF-1α) plays a key role in the pathological processes of cancers. Previous studies have shown that nickel can exert its carcinogenic effects through the induction of HIF-1α accumulation. However, the molecular mechanisms have not been well understood. The c-Jun N-terminal kinase 1 (JNK1) is implicated in several physiological processes, including proliferation, apoptosis and differentiation, which are thought to be involved in several cancer development. JNK1 is also known to regulate the level of Hsp90/Hsp70. Our present study was conducted to investigate the role of JNK1 in the accumulation of HIF-1α induced by nickel. In current studies, we demonstrated that JNK1 is required for such an Hsp-dependent HIF-1α regulation. HIF-1α stabilization was impaired in JNK1−/− cells, and could be rescued by reconstitutional expression of JNK1 in either hypoxia or chemical-mimicked hypoxia conditions. Accordingly, HIF-1-dependent transcriptional activity and its downstream gene's expression were dramatically reduced in JNK1-deficient cells. We further disclosed that in JNK1−/− cells the low expression level of Hsp90/Hsp70 proteins affected the protective roles of these chaperones in maintaining newly synthesized HIF-1α stabilization, and forced expression of Hsp90 or Hsp70 in JNK1−/− cells showed a notable increase in HIF-1α stability compared with that of parental cells. Furthermore, our studies found that defective HDAC6 expression and subsequently increased Hsp90 acetylation could account for the reduction of Hsp90 chaperon activity in JNK1−/− cells. Our studies for the first time disclose a novel function of JNK1 in the modulation of HIF-1ααstabilization under nickel mimicked chemical hypoxia conditions through regulation of Hsp90/Hsp70 expression as well as HDAC6-mediated Hsp90 acetylation modification. This novel function may contribute to JNK1-mediated carcinogenic effects in response to nickel exposure.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4373.
Abstract 4385: Testosterone induces DNA damage signaling in response to oxidative stress in prostate cancer cells
Authors: Ide Hisamitsu , Jingsong Yu , Yan Lu , Muto Satoru , Horie Shigeo
Published: February 2011

Introduction: In prostatic tissues, DNA damage response is specially activated in prostatic intraepithelial neoplasias (PINs) that are generally regarded as precursors of prostate cancer. The process of oncogenic transformation leads to enhanced DNA damage and activates the checkpoint network as an inducible barrier against cancer progression. In this study we analyzed the effects of testosterone under oxidative stress in DNA damage response to understand the biological function and the network of this signaling.

Methods: We tested the expression level of PSA and the activation of DNA damage response proteins including ATM (ataxia-telangiectasia-mutated kinase), H2AX (histone H2AX variant) and Chk2 (checkpoint kinase2) in prostate cancer cell lines (LNCaP and PC-3) with various concentration of H2O2. Apoptosis is quantified by flow cytometry and PARP cleavage.

Results: H2O2 induces apoptosis and the phosphorylation of ATM, H2AX and Chk2 in LNCaP cells. ATM inhibitor Ku55933 can block the phospholyration of H2AX and Chk2, but induced the activation of alternative pathways, p38 and ERK. Activation of DNA damage response and PARP cleavage was increased by testosterone. H2O2 did not induce phosphorylation of ATM, CHK2 and H2AX in PC-3 cells because of losing their expression. ATM inhibitor Ku55933 can reduce the apoptotic cells in LNCaP cells but PC-3 cells by the treatment of H2O2. Conclusion: Oxidative stress has been associated with tumor formation. On the other hand, DNA damage pathway plays important roles in the maintenance of the cell homeostasis in response to oxidative stress. Our results indicate that androgen signaling have tumor suppressive effects in prostate carcinogenesis through DNA damage response pathways under oxidative stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4385.
Abstract 4387: Reactive oxygen species-mediated cell death by psoralidin in prostate cancer cells
Authors: Sowmyalakshmi Srinivasan , Kumar Raj , Pahari Pallab , Rohr Jurgen , Damodaran Chendil
Published: February 2011

Modulating reactive oxygen species (ROS) levels may lead to either cell proliferation or death based on the cell type. It is well established that in cancer cells the basal levels of ROS is significantly higher when compared to their normal counterparts. Thus, identifying non-toxic small molecules which induces ROS specifically in cancer cells may be of enormous importance in cancer therapy. We have previously shown that psoralidin, a natural compound derived from Psoralea corylifolia specifically induces apoptosis in prostate cancer (PCa) cells (PC-3, DU-145, LNCaP and C4-2B). In this study we dissected the role of ROS in psoralidin-induced cell death in PCa. Our results suggested that psoralidin significantly induces ROS generation in both androgen-responsive (LNCaP) and -refractory (PC-3, DU-145 and C4-2B) PCa cells when compared to normal prostate epithelial cells (PzHPv7); leading to loss of mitochondrial membrane potential and release of cytochrome-c, caspase activation (9 and 3) and induction of apoptosis. In addition, induction of ROS downregulates pAkt expression and kinase activity, NF-κB (p65, p50 and p55) and mTOR (pp70S6K and p4E-BP1)-mediated pro-survival signaling in PCa cells. On the other hand, inhibition of ROS generation by N-acetylcysteine (NAC) prevented psoralidin-mediated anticancer effects on PCa. Collectively, our results suggest that psoralidin-mediated ROS generation in PCa cells causes inhibition of survival/proliferation and induction of apoptosis. Further investigation of the mechanism of action of psoralidin may facilitate initiation of clinical trials using psoralidin for the treatment of PCa.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4387.
Abstract 4388: beta-Carotene's oxidative breakdown inhibits p53 conformational folding and function
Authors: Bentz Brandon G. , Ring Terry A. , Murtaugh Maureen A. , Burnett David M. , McClane Robert , Fitzpatrick Frank A.
Published: February 2011

Lipophilic electrophiles have been found to have the ability to inhibit wild-type p53 protein function through a thioredoxin-dependent inhibition of functional protein folding. One potential extension of this line of research may help to explain the curious findings of certain carotenoid-based chemopreventive clinical trials that found that beta-carotene may be a contributory causal risk of cancer development in patients who actively smoke. Chemical precepts dictate that wild-type p53 function may be inihibited through a similar mechanism by other other lipophilic compounds such as carotenoids within a high redox milieu of ongoing smoking. We ask the question whether the oxidative breakdown of beta-carotene could inhibit p53 function via a similar post-translational mechanism. Using resonance Raman spectroscopy, HPLC, spectrophotometry, and Western blot analysis, we exposed isolated cell-free and intracellular beta-carotene to various oxidant sources such as nitric oxide (NO·), superoxide (O·), and/or the combination of NO· and O· [i.e. SNAP (NO+ donor), Spermine-NONOate (NO. donor), and SIN-1 (NO·/O· donor), and Angeli's salt (an NO[[Unsupported Character - Codename ­]]- donor)] as a model of ongoing oxidative stress. Our results demonstrate that the oxidative breakdown of beta-carotene specifically by SIN-1 (the donor of NO·/O· causes stabilization of p53, attributable to the accumulation of both “functional” and “non-functional” conformational folding. Finally, we demonstrate that the oxidative breakdown products of beta-carotene can inhibit thioredoxin function, thus providing a biochemical mechanism for the inhibition of wild-type protein folding. Taken together, these data support the hypothesis that ongoing oxidation of beta-carotene can inhibit this key cancer-related molecular pathway, through the post-translational inhibition p53 folding and function. These data may provide one biochemical mechanism for the surprising results of initial carotenoid-based chemopreventive cancer trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4388.
Abstract 4395: SEA technology: A novel strategy for enhancing antibody effector function
Authors: Alley Stephen C. , Sussman Django R. , Jeffrey Scott C. , Torrey Lindsay , Burke Patrick J. , Setter Jocelyn , Gfeller Brian , Klussman Kerry , Okeley Nicole M. , Senter Peter D. , Benjamin Dennis R.
Published: February 2011

The activity of monoclonal antibodies (mAbs) can be enhanced by a number of chemical and genetic strategies. We describe a novel strategy, Sugar Engineered Antibody (SEA) technology, for enhancing antibody-dependent cellular cytotoxicity (ADCC) through modification of the mAb carbohydrate. A series of small molecule fucose analogs were added to mAb-expressing Chinese hamster ovary (CHO) cells, with the resulting mAbs showing a significant reduction in their carbohydrate fucosylation. We demonstrate that these mAbs show markedly increased ADCC activity and improved CD16 binding. The fucose analogs inhibit GDP-mannose dehydratase (GMD), the first enzyme in de novo synthesis of GDP-fucose, and lead to global depletion of intracellular GDP-fucose. We also demonstrate that this strategy yields mAbs with significantly reduced fucosylation in large scale CHO cell culture and is broadly active across a variety of mAbs and expression systems. Since genetic modification of the mAb-producing cell line is not required, SEA technology can be readily applied from the mAb screening to manufacturing stage to generate effector function enhanced therapeutic antibodies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4395.
Abstract 4396: Conjugation site modulates the stability and biological activity of antibody drug conjugates
Authors: Shen Ben-Quan , Xu Keyang , Liu Luna , Parsons Kathryn , Bhakta Sunil , Raab Helga ,   20 more Genentech Authors, Flagella Kelly , Spencer Susan D. , Kaur Surinder , Scheller Richard H. , Sliwkowski Mark X. , Polakis Paul , Junutula Jagath Reddy
Published: February 2011

Antibody drug conjugates (ADCs) are attractive targeted chemo-therapeutic molecules as they combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity. The successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency and mode of linker-drug conjugation to the antibody. Recently, we have developed antibodies with cysteine substitutions (THIOMABs) at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., Nature Biotech., 26, 925-932, 2008). These THIOMABs can then be derivatized through the engineered cysteine thiol group to obtain ADCs with uniform stoichiometry (∼2 drugs per antibody). Studies with multiple antibodies against different antigens have shown that THIOMAB drug conjugates (TDCs) are as efficacious as conventional conjugates in xenograft models and are tolerated at higher doses in relevant preclinical models. To further understand improved therapeutic activity and in vivo metabolism of TDCs, we have engineered TDCs with drug attachment at different parts of the antibody (light chain-Fab, heavy chain-Fab and heavy chain-Fc). TDCs produced by THIOMAB technology provided an unique advantage over conventional ADCs to answer several unresolved questions in ADC therapeutics due to their homogeneity and site-specific conjugation to cytotoxic drugs. We will discuss novel findings that were observed with these TDC varaints and the influence of conjugation site in modulating stability and biological activity of antibody drug conjugates.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4396.
Abstract 4397: Development of antibody-drug conjugate technology based on novel linkers & DNA alkylating duocarmycin derivatives
Authors: de Groot Vincent F.M.H , Beusker Patrick , Joosten John , Spijker Henri , Coumans Ruud , Menge Wiro , Elgersma Ronald
Published: February 2011

Antibody-Drug Conjugation (ADC) is gaining momentum as a next generation antibody therapeutics approach in oncology. Tumor specific delivery of the cell-killing agent maximizes the agent's potency while avoiding damage to healthy tissue resulting in a high therapeutic window. Syntarga's Potent Payload Technology comprises the combination of highly potent DNA-alkylating duocarmycin derivatives and suitable linker technologies. Duocarmycin analogs and the CC-1065 derivatives represent a class of highly potent, cell-killing, DNA-alkylating, minor groove binding agents. This class is suitable to target solid tumors and has not been associated with Multi-Drug Resistance (MDR). Strong in vivo Proof of Concept with Potent Payload ADCs has been obtained against multiple targets. In human tumor xenograft models, substantial efficacies have been obtained based on single dose treatments, with minimal or absent side effects. We discuss applicability and several other aspects of multiple, validated Potent Payload-based Antibody-Drug Conjugates (ADCs). Latest results of preclinical development progress (in vivo efficacy, safety, etc.) will be presented, including drug potencies, linker stability and therapeutic window aspects for ADCs directed against HER2.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4397.
Abstract 44: STAT1-dependent expression of genes and proteins involved in energy metabolism links tumor growth and radioresistance to the Warburg effect and predicts poor prognosis in cancer patients
Authors: Khodarev Nikolai N. , Pitroda Sean P. , Wakim Bassam T. , Golden Daniel W. , Beckett Michael A. , Mauceri Helena J. , Sutton Harold G. , Perakis Samantha , Malik Renuku , Wietholt Christian , Pelizzari Charles , Chen Chin-Tu , Weichselbaum Ralph R.
Published: February 2011

Signal Transducer and Activator of Transcription 1 (STAT1) has traditionally been regarded as a transmitter of interferon signaling and a pro-apoptotic tumor suppressor. Recent data have identified new functions of STAT1 associated with tumorigenesis and resistance to genotoxic stress, including ionizing radiation (IR) and chemotherapy. To investigate the mechanisms contributing to the tumorigenic functions of STAT1, we performed a combined transcriptomic-proteomic expressional analysis of tumor xenografts with stable Stat1 knock-down (KD) relative to tumors with wild type (wt) Stat1. We also compared previously described SCC61 and nu61 isologous tumors, where SCC61 is radiosensitive and nu61 is radioresistant counterparts with different expression of IFN/Stat1 pathway. Transcriptional profiling was based on Affymetrix Human GeneChip® Gene 1.0 ST microarrays. Proteomes were determined from the MS/MS data by searching against the human subset of the UniProt database. Knockdown of STAT1 led to significant growth suppression in untreated tumors and radiosensitization of irradiated tumors. These changes were accompanied by alterations in the expression of genes and proteins of glycolysis/gluconeogenesis (GG), the citrate cycle (CC), and oxidative phosphorylation (OP). Of these pathways, GG had the most concordant changes in gene and protein expression and demonstrated a STAT1-dependent expression of genes and proteins consistent with tumor-specific glycolysis. IR drastically suppressed the GG pathway in STAT1 KD tumors without significant change in STAT1 WT tumors. Using 18F-FDG-PET we observed significantly higher glucose uptake in nu61 compared to SCC61 post-irradiation (6Gy). The STAT1 and glycolytic pathways were co-expressed in human breast tumors, and expression of STAT1-linked glycolytic genes was highly predictive of poor prognosis. Our results identify a previously uncharacterized function of STAT1 in tumors: expressional regulation of genes and enzymes involved in glycolysis, the citrate cycle, and mitochondrial oxidative phosphorylation, with predominant regulation of glycolysis. STAT1-dependent transcriptional and translational regulation of glycolysis suggests a potential role for STAT1 as a transcriptional modulator of genes responsible for the Warburg effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 44.
Abstract 440: Myoglobin in breast cancer: Regulation, function and relevance
Authors: Kristiansen Glen O. , Hu Junmin , Gerhardt Josefine , Fritzsche Florian , Theurillat Jean-Phillipe , Geisler Cordelia , Rose Michael , Stiehl Daniel , Gleixner Eva M. , Hankeln Tom , Gnaiger Erich , Moch Holger , Gassmann Max , Gorr Thomas , Dahl Edgar
Published: February 2011

Background

Following an accidental observation, we aimed to analyse expression, regulation and function of myoglobin (Mb) in breast cancer.

Methods

Immunohistochemistry/Immunofluorescence, microdissection, transmission electron microscopy, Western blot, qPCR, cell culture (under normoxia and hypoxia), shRNA and siRNA knockdown, in silico data mining, high resolution respirometry. Functional assays: proliferation, migration and colony formation.

Results

Mb protein is de novo expressed in 71% of invasive breast carcinomas (n=917) in association with the hypoxia inducible factor 2α (HIF-2 α), carbonic anhydrase IX and a better prognosis. Expression of Mb in breast cancer was confirmed on mRNA level and occurred irrespective of rhabdomyoid differentiation and was inducible by prolonged hypoxia in breast cancer cell lines (MDA-MB231, MDA-MB468, MCF7) with a median change fold of 3.4 after 72 hours. Interestingly, hypoxically induced MB mRNA originated from a novel, alternative transcription start site 6 kb upstream of the genuine ATG codon. Functionally, stable myoglobin knockdown in MDA-MB468 cells was associated with a stimulated O2 uptake during mild hypoxia, yet did not impact the O2 diffusion gradient in these small cells during limiting conditions. Knockdown of Mb also conferred a significant retardation of the cells’ in vitro motility to close an inflicted wound within the normoxic culture.

Conclusions

Regarding cancer cells, unconventional functions of myoglobin, not directly related to the transport of oxygen, thus need to be envisioned. These unprecedented findings may have fundamental implications for our understanding of non-muscle Mb in solid tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 440.
Abstract 4405: Orthogonal detection of thymidine analogue based cell proliferation and apoptosis using click chemistry combined with antibody based progesterone receptor detection in estradiol treated rat model
Authors: Clarke Scott T. , Arumugam Arunkumar , Lakshmanaswamy Rajkumar
Published: February 2011

Detection reagents which can simultaneously measure cell proliferation and apoptosis, or measure chronological changes in cell proliferation over the course of a treatment provide insight into efficacy and mechanisms of treatment on cell cycle and cell heath. Detection reagents which can temporally separate cell proliferation while combined with apoptosis detection provide a powerful new investigative tool. When 5-ethynyl-2′-deoxyuridine (EdU) incorporation is used in conjunction with bromodeoxyuridine (BrdU), cell proliferation measurements are made with orthogonal detection platforms permitting unambiguous identification of proliferation events. In rat mammary epithelial tissue changes in cell proliferation as a result of treatment combined with apoptosis detection demonstrates the value of click chemistry when combined with antibody based detection methods.

We use the rat model to show changes in proliferation and apoptosis rates in mammary epithelial tissue, after alteration in hormone treatment. Use of click chemistry labeling in ovariectomized rats shows a decrease in proliferation rate from their basal levels, while estradiol treatment results in an increase in proliferation rate over basal level. Cessation of a 7 day estradiol treatment causes a drop in proliferation rate and an increase in apoptosis which can be efficiently detected using click chemistry. Progesterone receptor levels are measured simultaneously with proliferation rate when click chemistry based detection is combined with antibody based detection methods.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4405.
Abstract 4411: Novel internalizing human scFvs targeting pancreatic tumor cells residing in their natural tissue microenvironment
Authors: Zhu Xiaodong , He Jiang , Zhang Xiaoju , Liu Yue , Zheng Guixi , Feng Jinjin , Su Yang , Kim Grace , Tempero Margaret , Liu Bin
Published: February 2011

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a five-year survival rate of about 4%. Current diagnostic and therapeutic regimens are limited and ineffective. The goal of this project is to develop novel internalizing human antibodies that target pancreatic tumor cells residing within their natural tissue microenvironment, and possess the ability to translocate through the cell membrane to reach the inside of live tumor cells. Antibodies with these two properties, i.e., being able to target tumor cells in human tissue specimen and to penetrate live tumor cells, can in the future be used to develop minimally invasive imaging strategy for earlier detection and targeted therapy that requires efficient and specific accumulation of payloads inside tumor cells. To achieve this objective, we have selected a large naïve phage antibody display library that we have recently constructed on microdissected human PDAC tissue specimens. We have identified a panel of internalizing human single chain antibodies (scFvs) that target pancreatic tumor cells in situ residing in their natural tissue microenvironment. Immunohistochemistry analysis showed that these scFvs bound to human PDAC, thereby recognizing clinically represented tumor antigens as opposed to cell line artifacts. All antibodies studied are internalized by PDAC cells in vitro. Using single photon emission computed tomography / computed tomography (SPECT/CT), we showed that technetium-labeled scFvs target human xenograft PDAC cells in vivo. Interestingly, a subset of these scFvs also target murine pancreatic tumor cell lines derived from transgenic PDAC mice, indicating that they target tumor epitopes conserved between human and mouse. Antibodies targeting tumor antigens across species would allow future imaging and therapy studies to be performed not only in xenograft but also transgenic PDAC models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4411.
Abstract 4414: EC0531: A potent folate-targeted tubulysin conjugate
Authors: Leamon Christopher P. , Reddy Joseph A. , Wang Yu , Dorton Ryan , Westrick Elaine , Bloomfield Alicia , Vetzel Marilynn , Klein Patrick J. , Vlahov Iontcho R.
Published: February 2011

Our lab is focused on the discovery and development of highly potent chemotherapeutic agents that target folate receptor (FR) expressing malignancies. One such compound, EC0305, represents a folate-tubulysin B construct capable of specifically eradicating well-established, FR-positive subcutaneous tumors from mice [Cancer Res (2008) 68:9839]. Recently, multiple polar carbohydrate segments (e.g. 1-amino-1-deoxy-glucitolyl–glutamate) were placed in-between the folate and tubulysin B moieties (i.e. the spacer region) of EC0305 to create a new compound with more desirable biological properties. When tested in culture, this novel agent, herein referred to as EC0531, was found to specifically inhibit the growth of FR-positive cells (IC50 of ∼ 2 nM) in a dose-dependent manner. Its cytotoxic activity was blocked in the presence of an excess of folic acid, and no activity was observed against FR-negative cells, thereby demonstrating this compound's target specificity. EC0531′s potency was confirmed againstin a human KB xenograft-nu/nu mice cancer model. Here, EC0531 therapy displayed remarkable anti-tumor activity (5/5 cures) with dose levels that did not cause weight loss or major organ tissue degeneration. In contrast, the anti-tumor activity was completely abolished (0/5 remissions) in EC0531-treated animals that were co-dosed with a benign folate-containing analog. The advantage provided by folate conjugation was further proven by the un-targeted free drug, which was found to be completely inactive (0/5 remissions) at both tolerable and highly toxic dose levels. Collectively, these results show that this potent anti-proliferative tubulysin compound can be specifically delivered to FR-positive tumors to provide substantial therapeutic benefit using well tolerated dosing regimens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4414.
Abstract 4418: ENMD-1198, a new microtubule-targeting agent with potent anti-vascular properties
Authors: Pasquier Eddy , Sinnappan Snega , Munoz Marcia , Kavallaris Maria
Published: February 2011

The formation of a new vascular network, by a process called angiogenesis, is a key driver in tumor growth and metastasis, making this an attractive therapeutic target in the fight against cancer. Different strategies are being developed to either prevent tumor angiogenesis (anti-angiogenic therapy) or disrupt the tumor vasculature already in place (vascular-disrupting therapy). ENMD-1198 is a new analogue of 2-methoxyestradiol (2-ME2), a microtubule-targeting agent that has shown promising results in the treatment of multiple myeloma, hormone refractory prostate cancer and recurrent ovarian cancer. Pre-clinical in vivo studies have shown that ENMD-1198 displayed improved metabolic stability as compared to its parental compound (2-ME2) and retained potent anti-tumor activity [1, 2]. We hypothesized that ENMD-1198 may also exhibit improved anti-angiogenic and/or vascular-disrupting properties. Using both bone-marrow derived and dermal microvascular endothelial cell lines, we thus analyzed and compared the effects of ENMD-1198 and 2-ME2 on the different functions of endothelial cells involved in angiogenesis. In both cell lines, ENMD-1198 was more potent than 2-ME2 at inhibiting endothelial cell proliferation, motility, migration and morphogenesis. In addition, videomicroscopy experiments revealed that ENMD-1198 was able to completely disrupt pre-formed vascular structures within 2 hours. This vascular-disrupting activity was associated with extensive depolymerisation of microtubules, accumulation of actin stress fibers and alteration in focal adhesions. Collectively, our results demonstrate that this new compound displays improved anti-vascular properties and this study provides important insights into the mechanism of action of ENMD-1198, a promising new anti-cancer drug.

1. LaVallee T.M, Burke P.A, Swartz G.M et al. (2008) Mol Cancer Ther.7: 1472-82.

2. Moser C., Lang S.A., Mori A. et al. (2008) BMC Cancer 8:206.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4418.
Abstract 4426: Are there synergistic antiproliferative effects of paclitaxel and curcumin on osseus metastasis of renal cell carcinoma
Authors: Reinecke Petra , Biewer Matthias , Kalinski Thomas , Gabbert Helmut Erich
Published: February 2011

Aim: Human renal cell carcinomas (RCCs) are highly resistant to conventional chemotherapy. Recently, paclitaxel has been shown to inhibit growth of RCCs in vitro. Therefore, we analyzed the chemotherapeutic potential of paclitaxel on our established cell line N109, derived from an osseous metastasis of a human RCC. In addition, we examined the role of the phytochemical curcumin on the growth inhibition which is known to act as a modulator of the Multidrug Resistance Phenotype. Methods: MTT assay, immunfluorescence microscopy and flow cytometry. Results: 1. Significant dose-dependent growth inhibition and paclitaxel-induced morphological alterations were observed in the cell line N109. 2. No significant dose-dependent growth inhibition was observed after exposure to curcumin alone. 3. A synergistic effect of growth inhibition was detected after treatment with both, paclitaxel and curcumin. 4. The cell line N109 shows a pronounced P-glycoprotein expression. 5. After exposure to paclitaxel or curcumin no remarkable modulation of the P-glycoprotein expression level was observed. Conclusion: This study demonstrates that paclitaxel can effectively inhibit the growth of an osseous metastasis of human renal cell carcinoma in vitro whereas a pronouced synergistic inhibition effect in combination with the phytochemical curcumin became evident.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4426.
Abstract 4428: A novel imidazo[4,5-b]pyridine Aurora kinase inhibitor with anti-leukemic activity
Authors: Moore Andrew , Bavetsias Vassilios , Sun Chongbo , Faisal Amir , Workman Paul , Blagg Julian , Pearson Andrew , Linardopoulos Spiros
Published: February 2011

The Aurora kinases are a family of highly conserved serine-threonine kinases that play key roles in several stages of mitosis. Over-expression of these kinases has been demonstrated in a range of malignancies including leukemia. Aurora kinase inhibitors are emerging as promising agents in the treatment of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The activity of this class of drugs in AML and CML may be in part due to their ability to also inhibit the tyrosine kinases FLT3 and Abl1 respectively. Here we report the activity of a novel imidazo[4,5-b]pyridine derivative developed at our institution with potent inhibition of Aurora A and B kinases (IC50 values of 6 nM and 45 nM respectively). Furthermore, the IC50 for FLT3 kinase is < 0.5 nM, whilst the IC50 for Abl1 kinase is 82 nM. Consistent with the potent kinase inhibition profile, the AML cell lines MV-4-11 and MOLM-13 are particularly sensitive with GI50 values less than 50 nM. Cellular assays demonstrate that this imidazo[4,5-b]pyridine derivative inhibits phosphorylation of histone H3, a direct target of Aurora B kinase, as well as phosphorylation of FLT3 and the downstream p44/42 MAP kinases in these cell lines. Both MV-4-11 and MOLM-13 carry internal tandem duplications of the FLT3 receptor tyrosine kinase (FLT3-ITD), which is clinically associated with a poor prognosis in AML. The potent preclinical activity in FLT3-ITD positive cell lines supports the growing body of evidence that dual pan-Aurora and FLT3 kinase inhibitors may be of benefit in the high-risk group of patients with FLT3-ITD positive AML.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4428.
Abstract 4429: Characterization of the kinesin spindle protein inhibitor AZD4877
Authors: Pinzon-Ortiz Maria C. , Cao Alex , Sheehy Adam , Pablo Lourdes , McEachern Kristen , Hylander-Gans Linda , Wu Kaida , Reimer Corinne , Morosini Deborah , McCoon Patricia , Huszar Dennis
Published: February 2011

The process of mitosis is a validated point of intervention in cancer therapy and a variety of anti-mitotic drugs are successfully being used in the clinic. To date, all approved antimitotics target the spindle microtubules, thus interfering with spindle dynamics, leading to mitotic arrest and apoptosis. While effective, these drugs are also associated with a variety of side effects, including neurotoxicity. In recent years, mitotic kinesins have attracted significant attention in the search for novel, alternative mitotic drug targets. In addition, kinesin inhibitors may overcome resistance to microtubule targeting drugs.

Here we describe the pharmacological evaluation of AZD4877, a selective Eg5 inhibitor. AZD4877 exhibits potent cell growth inhibition in vitro against a broad panel of both solid and hematological tumor cell lines. Efficacy is associated with formation of monoastral spindles, mitotic block and the induction of apoptosis. AZD4877 also demonstrates activity in multiple tumor xenograft models, including primary bladder tumor explants and a Rituximab-insensitive non-Hodgkin's Lymphoma model (DoHH2T53). Combination studies with a taxane agent (Docetaxel) demonstrate additive efficacy of tumor growth inhibition, with notable tumor regrowth delay in the combination arm. In summary, AZD4877 is an active Eg5 inhibitor with potent anti-tumor activities in vitro and in vivo. The efficacy observed in combination studies and in a rituximab-resistant tumor model suggests possible clinical applications for this agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4429.