bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4059: Identifying the key domain of CHK1 interaction with MEPE/OF45 and the key ubiquitination site of CHK1
Authors: Chen Guangnan , Yan Dan , Wang Ya
Published: August 2014

Background: Previously, we reported that MEPE/OF45 interacting with CHK1 protects CHK1 from degradation and cells from DNA damage-induced killing (Nucleic Acids Res, 37: 7447-7454 2009; Brit J Cancer 102: 862-866, 2010). To further investigate the nature of the CHK1 stabilization and its effects on cell sensitivity to DNA damage inducers, we designed this study to identify the key domain of CHK1 interaction with MEPE/OF45 and the key ubiquitination site of CHK1.

Methods: GST-MEPE/OF45 was expressed in E. coli BL 21. HA-wild type CHK1 and various CHK1 truncated mutations were expressed in 293FT. GST pull-down assay was used to identify the key domain of CHK1 interacting with MEPE/OF45. To identify the ubiquitination site of CHK1, GFP-CHK1 with different Lys mutations were co-transfected with Ub-expression vector to 293FT, and 48 hours later, whole cell lysate were prepared, Western blot was carried out using a GFP monoclonal antibody to detect ubiquitinaed CHK1. The CHK1 half-life was used to detect the stabilization of CHK1 and a clonogenic assay was used to detect the sensitivity of cells to DNA damage inducers.

Results: The key domain of CHK1 interacting with MEPE/OF45 and the key site of CHK1 ubiquitination were identified. Most importantly, the mutation of CHK1 with the main domain or key site affected CHK1 stabilization and cell sensitivity to DNA damage inducers.

Conclusion: The interaction of CHK1 with MEPE/OF45 plays an important role in preventing CHK1 ubiquitination and; therefore, stabilizes CHK1, which further affects cell sensitivity to DNA damage inducers. Because CHK1 is an essential DNA damage response protein, these results provide valuable information for understanding the role of CHK1 in genomic integrity, which could be used for improving cancer treatment.

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 4059. doi:10.1158/1538-7445.AM2011-4059
Abstract 4060: Enriching for rrare subpopulations of circulating microvesicles by the depletion of endothelial- and leukocyte-derived microvesicles
Authors: Spetzler David B. , Deng Ta , Tinder Teresa , Kimbrough Jeff , Ellis Phil , Holterman Daniel , Maney Todd , Kuslich Christine D.
Published: August 2014

Endothelial- and leukocyte-derived circulating microvesicles (cMV) represent a majority of the cMV present in the blood. The purpose of this study was to determine if depletion of these more common cMV populations is possible, allowing for the enrichment and analysis of the remaining subpopulations of cMV. Circulating microvesicles were isolated from the blood plasma of breast cancer patients. Magnetic beads conjugated to antibodies specific for either CD31 or CD45 were used to deplete the isolated endothelial- and leukocyte-derived cMV. The remaining cMV population was characterized with a multiplexed immunoassay against a panel of 20 different antigens. In addition to the expected depletion of the biomarker used to remove the targeted cMV in the remaining cMV population, a concomitant depletion of associated biomarkers was observed. In the case of the cMV population in which CD31 had been used to remove endothelial-derived cMV, for instance, there was a significant concomitant depletion of DLL4, a highly associated endothelial marker. This was not the case with general epithelial microvesicle markers, such as CD9, which were still very much present in the cMV population remaining after depletion. These findings suggest a potential method for a more complete characterization of biomarker profiles associated with cMV derived from disease-associated cells and provide the foundation for a novel cMV-based strategy for disease detection through a noninvasive blood test.

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 4060. doi:10.1158/1538-7445.AM2011-4060
Abstract 4134: Exploration for possible predictive biomarkers for individual response to mFOLFOX6 in colorectal cancer patients
Authors: Suzuki Okihide , Murata Kohei , Fukunaga Mutsumi , Takemoto Hiroyoshi , Ohue Masayuki , Ikeda Rikuko , Wada Satoru , Eguchi Hidetaka , Tomita Naohiro , Watanabe Masahiko , Ishida Hideyuki , Nishiyama Masahiko
Published: August 2014

Background: Incorporation of molecular targeting agents such as bevacizumab, cetuximub, and panitumumab, into cytotoxic chemotherapeutic regimens has increased survival in colorectal cancer patients, resulted in provision of a promising means of individualization of these therapies. Nonetheless, the clinical efficacy and toxicity of basic regimens, such as FOLFIRI and FOLFOX, remain unpredictable. Thus, identification of molecular indicators of individual response to the basic regimens is urgently required. Methods: This prospective study was conducted to determine potent predictive markers of individual response to mFOLFOX6, in terms of efficacy and toxicity. Chemonaive patients with stage IV colorectal cancer were eligible for this trial after a palliative operation, and received mFOLFOX6 treatment. Along with a search for KRAS gene somatic mutations, analyses of mRNA expression levels in cancerous portions were conducted for 10 candidate marker genes, namely DPYD, TYMS, ERCC1, ERCC2, XRCC1, GSTP1, EGFR, VEGF, TYMP and TNFRSF1B, by means of real-time RT-PCR. In addition, some acknowledged polymorphisms of DPYD, TYMS, ERCC1, ERCC2, XRCC1, GSTP1 and EGFR genes were also determined. Results: Fifty pts received a total of 424 treatment cycles. Overall response rate was 60.4% (1CR and 28 PRs), and median overall and progression survival were 930 days and 234 days, respectively. The most common grade 3/4 toxicity was neutropenia (40%), followed by anorexia (6%), anemia (4%), leucopenia (4%), etc. The therapeutic response was not related to KRAS somatic mutational status, and no genotype-toxicity association was observed, thus far tested. On the other hand, ERCC1, ERCC2, and TYMS gene expression levels were significantly higher in patients with PD than in those with CR/PR/SD (P=0.017, 0.028, and 0.032, respectively). The expression levels of TYMS were highly correlative with best tumor response rate (R=0.414, P=0.004). Overall survival was significantly better in patients with low ERCC2 expression (P=0.039). Furthermore, combined expression levels of ERCC1, ERCC2, and TYMS were found to be associated with PFS: Those patients with high expression levels of all of these 3 genes showed poorer survival than the others (P=0.0029). Conclusions: Expression levels of ERCC1, ERCC2, and TYMS genes in tumor may be predictable for individual response to mFOLFOX6. A large-scale prospective study to validate their predictive values is now under planning.

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 4134. doi:10.1158/1538-7445.AM2011-4134
Abstract 4156: Detection of lung cancer from plasma using the biosignature of circulating microvesicles
Authors: Tasinato Andrea , Hassaine Gherici , Ristorcelli Elodie , Chen Wang-Juh , Kuslich Christine D.
Published: August 2014

Circulating microvesicles (cMV) are cell-derived structures that are abundantly present in the blood. Tumor cells produce large quantities of cMV, and their abundance has been shown to correlate with tumor invasiveness and resistance to therapy. This study attempts to understand the origin, composition, and potential clinical utility of cMV, analyzing their protein composition in patients with non-small cell lung cancer (NSCLC) and identifying a cMV-based biosignature that can be used to predict the presence of tumors from a blood sample.

cMV were isolated from plasma samples that were obtained from an initial cohort of 65 patients with NSCLC and from 46 control samples. A discovery panel of 63 specific biomarkers was used to develop an assay with high specificity and sensitivity based on 4 cMV surface proteins. Using a novel cMV-based multiplexed analysis platform we optimized a threshold level for 4 significant cMV subpopulations to effectively distinguish lung cancer patients from controls.

The multiplex assay included 1 general cMV marker (CD81) and 3 lung cancer-associated biomarkers: the lung epithelial C-type lectin members SPD and SPA, plus the sialoprotein osteopontin. The decision tree analysis, based on specific cut offs, showed a sensitivity of 81% and a specificity of 90%.

These results provide initial evidence that the identification of biosignatures in distinct subpopulations of cMV may offer a powerful blood-based approach for the detection and monitoring of specific disease states, such as NSCLC.

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 4156. doi:10.1158/1538-7445.AM2011-4156
Abstract 4161: Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine
Authors: Millholland John M. , Patel Sheryl G. , Fernandez Cecilia A. , Shuber Anthony P.
Published: August 2014

Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary Matrix Metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine.

Methods: Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. These LNA suppress wild-type DNA amplification.

To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform.

Results: To validate the new the multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats.

Conclusions: By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance.

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 4161. doi:10.1158/1538-7445.AM2011-4161
Abstract 417: α9β1 integrin, as a critical regulator of lymphatic metastasis and growth of human breast cancer cells
Authors: Ohta Daichi
Published: August 2014

Osteopontin (OPN) has been implicated in various stages of cancer progression such as malignant transformation, invasion and metastasis. Several groups independently demonstrated that plasma OPN level is a potential diagnostic and prognostic marker for several human malignancies (Bellahcene A et al, Nat Rev Cancer 8:212, 2008 and Anborgh PH, et al Clin Chem 55:895, 2009). Interestingly, a human breast cancer cell with an aggressive lymph node metastatic ability has been established, which demonstrated strong expression of α9β1 integrin and OPN (Vantyghem SA et al Clin & Exp Meta 22:351, 2005). Of note, α9β1 integrin is a receptor for not only OPN, but also VEGF-C and -D, known lymphatic vessel growth factors. To address whether and how α9β1 integrin is involved in tumorigenesis, growth, invasion and lymph node metastasis, we took advantage of using an inhibitory anti-human α9 integrin antibody (clone: K33N), which does not cross react with murine α9 integrin and an α9β1 integrin-positive human breast cancer cell line, MDA-MB-231 luc-D3H2LN (D3H2LN), in in vitro functional assays and an in vivo orthotopic xenotransplantation model.

We found that K33N inhibited in vitro migration and invasion of D3H2LN cells, indicating that the interaction of α9β1 integrin with its ligands was involved in the breast cancer cells motility. Next, we inoculated D3H2LN cells into the mammary fat pad of nude mice at day 0, and K33N or control IgG was given to them twice per week from day 15 to day 50 after tumor inoculation. K33N significantly reduced both primary tumor growth and draining lymph node metastasis. To gain an understanding of the mechanism by which K33N suppressed the primary tumor growth and lymph node metastasis, we examined the expression of various molecules which are associated with the cancer progression. We found significant matrix metalloproteinase (MMP) up-regulation in K33N-treated primary tumor tissues, consistent with a recent report demonstrating an indirect involvement of MMPs involved in tumor growth. Of note, upon orthotopic injection of human D3H2LN cells, plasma level of host-derived murine OPN, but not tumor-derived human OPN was significantly elevated. Surprisingly, host-derived murine OPN was significantly reduced in the mice treated with K33N. These results suggest that the interaction between α9β1 integrin on breast cancer cells and its ligands favors the generation of tissue microenvironments which contribute to primary tumor growth and lymph node metastasis.

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 417. doi:10.1158/1538-7445.AM2011-417
Abstract LB-200: Solution structure of c-Myc G-quadruplex complex with a quindoline compound
Authors: Dai Jixun , Carver Megan , Mathad Raveendra I. , Hurley Lawrence , Yang Danzhou
Published: August 2014

Overexpression of the c-Myc oncogene is associated with a broad spectrum of human cancers. Targeting transcriptional control of c-Myc represents an attractive means for cancer molecular therapeutics. A highly conserved nuclear hypersensitivity element III1 (NHE III1) of the c-Myc promoter controls 80–90% of the c-Myc transcription. It can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-Myc transcriptional silencing. Small molecules that stabilize the c-Myc G-quadruplex have been shown to suppress c-Myc transcription and are anti-tumorigenic. We have determined the NMR structure of a 2:1 complex of the c-Myc quadruplex with a quindoline compound. This is the first molecular structure of a drug complex with a natural oncogene promoter quadruplex. The solution structure of this 2:1 quindoline-G-quadruplex complex shows unexpected features, including the drug-induced reorientation of the flanking sequences to form a new binding pocket. While both 3′ and 5′ complexes show overall similar features, there are identifiable differences that emphasize the importance of both stacking and electronic interactions. We identified the importance of the two flanking bases as well as the shape of the ligand in determining drug-binding specificity. The structural information will be crucial for structure-based rational drug design of molecules that specifically target the c-Myc silencer element. It also provides important implications for rational design of drugs that bind to unimolecular parallel G-quadruplexes commonly found in promoter elements.

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 LB-200. doi:10.1158/1538-7445.AM2011-LB-200
Abstract LB-201: New derivatives of etoposide show promise as more potent inhibitors of cancer cell growth
Authors: Gal Susannah , Young Rebecca , Slilaty Steve , Deliencourt-Godefroy Geraldine , Quirion Jean-Charles
Published: August 2014

The DNA repair enzyme Topoisomerase II (TopoII) has been recognized in the oncology field as a clinically important therapeutic target for a variety of cancers. The class of agents known as etoposides were specifically designed with this target in mind. Etoposides lacked the major problem of cardiac toxicity associated with the anthracyclines class of drugs (e.g. doxorubicin).

Sunshine Biopharma has recognized that the reduced efficacy and high toxicity of etoposide are due to instability of the molecule leading to a conversion of the drug which then functions as a tubulin inhibitor rather than a TopoII inhibitor (unpublished observations). Accordingly, the company set out to develop a true TopoII inhibitor and these efforts have yielded two compounds called Adva-27a and Adva-32a. These drugs and their derivatives have been described in US Patent Application Number: 20090318675. Using cell lines derived from a variety of human cancers, Adva-27a is almost always a more potent inhibitor of cell growth compared to etoposide, and cells expressing the MDR-drug resistance protein are very sensitive to this drug.

Furthermore, consistent with the literature, amplification of the TOP2α gene is associated with increased sensitivity to growth inhibition. The preliminary data show that in all but the brain tumor cell line, increased TOP2α copy number is associated with even greater percent inhibition by Adva-27a versus etoposide regardless of the tissue of origin of the tumor cell lines. This data and plans for further analysis of these drugs will be presented.

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 LB-201. doi:10.1158/1538-7445.AM2011-LB-201
Abstract LB-212: XGFR, an Fc-engineered dual signaling inhibitor targeting IGF-1R and EGFR
Authors: Wartha Katharina , Croasdale Rebecca , Schanzer Juergen , Brinkmann Ulrich , Hofmann Marco H. , Ries Carola , Rieder Natascha , Hoelzlwimmer Gabriele , Freytag Olivier , Herter Sylvia , Gerdes Christian , Kuenkele Klaus-Peter , Klein Christian
Published: August 2014

Background: Elevated signaling via the receptor tyrosine kinases IGF-1R and EGFR has been identified as common characteristic of multiple cancer type. IGF-1R and EGFR signal predominantly through the PI3K and MAPK signaling pathways and thereby mediate growth and survival signals crucial for the development and progression of cancer. There is strong cross talk on multiple levels between IGF-1R and EGFR dependent signaling pathways. Therefore, targeting IGF-1R and EGFR simultaneously is an attractive way to achieve maximal inhibition of signal transduction and to avoid resistance formation.

Methods: Bispecific IGF1R-EGFR antibodies were engineered by linking scFv domains of an EGFR Mab (GA201) via Serine-Glycine linkers to an IgG1 IGF-1R Mab (RG1507). The functional properties of the bispecific antibodies were evaluated in cellular in vitro assays (IGF-1R/EGFR phosphorylation, downregulation, 3D proliferation and ADCC assays) and in in vivo xenograft models for tumor growth inhibition and survival.

Results: Bispecific IGF-1R-EGFR antibodies (XGFR2, XGFR3, XGFR4) were successfully generated with yields and stability comparable to conventional IgG1 antibodies. XGFR antibodies effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation in H322M tumor cells and induced strong downmodulation of IGF-1R and enhanced EGFR downmodulation compared to the parental EGFR antibody GA201. XGFR antibodies showed strong anti-tumor efficacy comparable to the combination of monospecific IGF-1R and EGFR Mabs in the BxPC3 and H322M xenograft models. To enhance the ADCC properties of XGFR, afucosylated, glycoengineered bispecific antibodies with enhanced affinity for FcγRIIIA were generated using the GlycoMab technology. Glycoengineered bispecific antibodies were shown to have superior ADCC properties in in vitro ADCC assays and XGFR4 significantly prolonged median and overall survival of mice in an ADCC competent in vivo model (A549 i.v.).

Conclusions: Bispecific IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components of multiple cancer types (IGF-1R and EGFR), resulting in effective inhibition of the PI3K and MAPK signaling pathway and to avoid the formation of resistance to therapy. Having overcome issues of stability and productivity, bispecific antibodies may become an advantageous way to reduce costs and infusion times in cancer therapy, while at the same time, achieving maximal anti-tumor effects through inhibition of multiple targets.

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 LB-212. doi:10.1158/1538-7445.AM2011-LB-212
Abstract LB-36: Circulating epithelial tumor cells are responsive to BMP-2
Authors: Bähring Franziska , Reber Elena , Müller Ute , Kilper Roland , Wagner Kathleen , Hochhaus Andreas , Clement Joachim H.
Published: August 2014

Aims: Bone morphogenetic proteins (BMP) play an important role in early embryonic development as well as in organ homeostasis and in tumorigenesis. We could show previously that BMP-2 enhance migratory and invasive properties of tumor cells. Furthermore, BMP-2 affects the expression of genes which are involved in the regulation of apoptosis. A crucial step in metastasis formation is the liberation of tumor cells from the primary site, the entrance into circulation and the long-lasting persistence in the peripheral blood. Therefore we asked whether BMPs might contribute to the vitality of circulating tumor cells.

Methods: Leukocytes and circulating epithelial cells from the peripheral blood of breast cancer patients were labelled with fluorescent antibodies and magnetically enriched with the MACS® system. After separation of single vital cells with the aureka® device, a single cell multiplex RT PCR was performed using the AmpliGrid system. The PCR products were analysed by urea-polyacrylamide gel electrophoresis. For real-time single cell PCR demonstrating the expression level of selected genes the Fastlane system (Qiagen, Hilden) was used. Localization of phosphorylated receptor Smads was analysed by laser scanning microscopy after one hour incubation with 100 ng/ml BMP-2.

Results: Circulating epithelial cells were identified by using the cell surface antigen EpCam. Vital EpCam-positive cells were picked and subjected to multiplex RT-PCR covering 5 components of the BMP signaling cascade as well as RPL13A for control. 121 of 265 selected cells expressed RPL13A. These cells were used for further analysis. Most of these cells expressed BMP-2 (54%), BMPR-IA (46%), BMPR-IB (82%). Transcripts of the type II receptor BMPR-II (35%) and the putative BMP antagonist BMP-3 (25%) were presen to a lower extend. The expression level of BMP-2 was comparable in individual EpCam-positive cells, thus at least an autocrine stimulation of the BMP signaling pathway can be assumed. For studying the responsiveness of circulating epithelial cells towards a BMP stimulus these cells were enriched from peripheral blood and incubated with BMP-2. Vital cells showed a predominant nuclear staining for pSmad1 demonstrating a functional BMP signalling network.

Conclusion: We show that key components of the BMP signaling pathway are expressed in leukocytes and epithelial cells in the peripheral blood of breast cancer patients. This indicates that the BMP signaling network potentially contributes to the activity and survival of circulating epithelial cells.

This work was supported by the Dr. Rainald-Stromeyer-Stiftung

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 LB-36. doi:10.1158/1538-7445.AM2011-LB-36
Abstract LB-363: DNA-damage foci persistence and accelerated senescence as determinants of radiosensitization by PARP inhibitors
Authors: Efimova Elena V. , Mauceri Helena J. , Barreto Juan C. , Labay Edwardine , Sikora Jacek , Takahashi Satoe , Wolfgeher Don W. , Bindokas Vytautas P. , Darga Thomas E. , Beckett Michael A. , Sutton Harold G. , Vokes Everett E. , Posner Mitchell C. , Weichselbaum Ralph R. , Kron Stephen J.
Published: August 2014

Along with apoptosis and necrosis, accelerated senescence may be a desirable outcome when replicative cancer cells undergo unrepairable DNA double strand breaks (DSBs) after treatment with ionizing radiation (IR) or chemotherapy. In both normal and cancer cells, DSBs promote the assembly of Ionizing radiation induced foci (IRIF), domains of modified chromatin that serve a key role in DNA damage signaling and DNA repair. IRIF persistence is a critical determinant of accelerated senescence, making targeting IRIF resolution an attractive strategy to sensitize cancer cells to genotoxic therapy. As an IRIF reporter, we constructed an inducible GFP fusion to the IRIF-binding domain of 53BP1 (GFP-IBD) which we have expressed in breast, head and neck, prostate, glioma and pancreatic cancer cell lines. Typically, within minutes after IR exposure, GFP-IBD relocalizes to form fluorescent nuclear foci, which disperse over several hours. Anti-GFP immunoprecipitation and proteomic analysis of IRIF revealed chromatin modifiers and DNA damage response proteins at DSBs including the poly(ADP-ribose) polymerase PARP1. To block DSB repair in cancer cells and tumors, we treated cells with veliparib (ABT-888), one of several PARP inhibitors currently in clinical trials. Veliparib markedly enhanced IRIF persistence and increased cell senescence in the majority of cancer cell lines tested. Treatment of tumor xenografts in nude mice with veliparib and IR induced persistent foci, increased senescence in tumor and stroma, and delayed tumor regrowth. Our results suggest that the efficacy of PARP inhibitors may partially depend on a competent senescence response to accumulated DNA damage, validating this response as a desirable outcome of genotoxic therapy.

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 LB-363. doi:10.1158/1538-7445.AM2011-LB-363
Abstract LB-365: Unfolded protein response regulator BIP/HSPA5 as a chemoresistance biomarker and molecular target in acute lymphoblastic leukemia
Authors: Uckun Fatih M. , Qazi Sanjive , Garner Amanda , Ma Hong , Ozer Zahide , Pitt Jason , Janda Kim
Published: August 2014

BIP/HSPA5, also known as glucose regulated protein (GRP) 78, is an endoplasmic reticulum (ER)-resident multifunctional molecular chaperone that represents a pivotal component of the pro-survival arm of the unfolded protein response (UPR) signaling pathway. We performed a comprehensive meta-analysis of 17 datasets and 668 leukemia/lymphoma cases using the Oncomine Clinical Research Database. 7 of 10 contrasts for high-grade B-lineage lymphoid malignancies exhibited signigficant up-regulation of BIP/HSPA5, including B-lineage acute lymphoblastic leukemia (ALL) (2-fold, P=5.9×10–10). Notably, the gene expression profiles of primary leukemia cells in matched-pair leukemic specimens obtained from 27 B-lineage ALL patients both at the time of initial diagnosis and at the time of relapse were extracted from the NCBI Gene Expression Omnibus (GSE18497) revealed a significant 1.4-fold up-regulation of the BIP8/HSPA5 gene in relapse clones (P=0.0016). Likewise, anti-BIP/HSPA5 Western blot analysis of whole cell lysates from primary leukemic cells from relapsed and newly diagnosed B-lineage ALL patients revealed higher levels of BIP/HSPA5 protein in relapse specimens. Confocal microscopy revealed abundant expression of BIP/HSPA5 protein in the plasma membrane of chemotherapy-resistant ALL cells. Pep42, a cyclic 13-mer cell-penetrating peptide that was identified by screening a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cells, was found to not only specifically bind to surface expressed BIP/HSPA5 but also enter BIP/HSPA5+ cells by receptor-mediated endocytosis. We examined the ability of a doxorubicin conjugate of Pep42 (PEP-DOX) containing a cathepsin B-cleavable linker to target and induce apoptosis in surface BIP/HSPA5+ chemotherapy-resistant B-lineage ALL cells. By using a quantitative flow cytometric apoptosis assay, we documented that within 48 hours of treatment with 0.1–1.0 μM PEP-DOX (but not unconjugated drug-free Pep42, which was included as a control) apoptosis is induced in ≥99.9% of surface BIP/HSPA5+ ALL cells. As PEP-DOX specifically targets surface expressed BIP/HSPA5, 100-fold molar excess of drug-free Pep42 was able to compete with PEP-DOX for surface BIP/HSPA5 binding sites and thereby prevent PEP-DOX-induced apoptosis. Excess recombinant EGF, that was included as a negative control, did not affect PEP-DOX- induced apoptosis. The identification of the BIP/HSPA5 as a chemoresistance biomarker and molecular target for B-lineage ALL may lead to new anti-leukemic treatment strategies for relapsed disease that are much needed.

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 LB-365. doi:10.1158/1538-7445.AM2011-LB-365
Abstract LB-381: Inhibition of the hedgehog signaling pathway has significant preclinical activity and prevents development of androgen-independent prostate cancer
Authors: Mahalingam Devalingam , Liang Sitai , Buonamici Silvia , Sun LuZhe
Published: August 2014

Introduction: Prostate cancer (PCa) is the second leading cause of cancer death in American men. Patients with advance PCa are often treated first with anti-androgen therapy, but will invariably develop androgen-independent prostate cancer (AIPC). A recent study has shown that androgen deprivation can lead to increased expression of hedgehog signaling components. Hedgehog signaling is also known for normal and cancer stem cell self-renewal. Therefore, in this study, we tested the hypothesis that the inhibition of hedgehog pathway with a novel hedgehog inhibitor, LDE225 (Novartis®), will block the development and growth of AIPC.

Methods and Results: Two androgen-dependent PCa (ADPC) cell lines were adapted to grow in an androgen-free culture medium. The emergence of AIPC cells was found to be associated with increased expression of hedgehog ligands, and hedgehog signaling activity through the activation of its downstream Gli transcription factors as detected using a Gli-responsive promoter luciferase reporter. We also found that treatment with LDE225 was more effective in inhibiting the growth of AIPC cells than ADPC cells. Furthermore, the treatment with LDE225 also inhibited the emergence of AIPC cells when the ADPC cells were cultured in the androgen-free medium. Finally, treatment of orthotopic xenograft PCa model with LDE225 is underway to confirm our pre-clinical in-vitro activity in both ADPC and AIPC models.

Conclusion: These results support our hypothesis that the activation of hedgehog pathway contributes to the development of AIPC and provide the rationale and guidance for design of clinical trials utilizing LDE225 in PCa patients.

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 LB-381. doi:10.1158/1538-7445.AM2011-LB-381
Abstract LB-401: The chemosensitizing properties of iniparib in combination with DNA-damaging agents in the MDA-MB-468(−) triple-negative breast cancer (TNBC) cell line
Authors: Ossovskaya Valeria , Lim Chang-Uk , Schools Gary , Kalurupalle Swathi , Roninson Igor B. , Broude Eugenia
Published: August 2014

BACKGROUND: Iniparib (BSI-201) has demonstrated promising efficacy and safety in the treatment of patients with metastatic triple-negative breast cancer (TNBC). The addition of iniparib to gemcitabine (G) and carboplatin (C) improved the clinical benefit rate from 34% to 56% (P = 0.01) and the overall response rate from 32% to 52% (P = 0.02) in a phase II clinical study (O'Shaughnessy et al. N Engl J Med. 2011).We investigated the chemosensitizing properties of iniparib with irinotecan (I) or ionizing radiation (IR).

METHODS: The chemosensitizing properties of a single dose of iniparib combined with I or IR were evaluated in TNBC MDA-MB-468 breast carcinoma cells. Cell proliferation was evaluated with FACS-based cell cycle analysis, including DNA staining and bromodeoxyuridine incorporation.

RESULTS: FACS analysis showed that in TNBC MDA-MB-468 breast carcinoma cells iniparib potentiated cell cycle arrest induced by I, with cells arrested in S and G2/M phases, or potentiated cell cycle arrest induced by IR, with cells arrested mostly in G2/M, at 72 hours after treatment (Table 1).

CONCLUSIONS: These data demonstrate the potentiation of the antiproliferative activity of I and IR by iniparib in TNBC MDA-MB-468 breast carcinoma cells. These data support investigation of iniparib in combination with these DNA-damaging agents in clinical studies.

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 LB-401. doi:10.1158/1538-7445.AM2011-LB-401
Abstract 1172: Phase I study of histone deacetylase inhibitor belinostat in combination with warfarin in patients with solid tumors or hematological malignancies.
Authors: Agarwal Neeraj , Wade Mark L. , Batten Julia , Davidson Cynthia , Sun Show-Li , Sharma Sunil
Published: August 2014

Background: The family of Histone Deacetylase (HDAC) enzymes serves as important epigenetic regulators of gene expression through modulation of acetylation on important histone and non-histone proteins. Aberrant acetylation of histones can alter gene expression believed to be important in the tumorigenic process. Belinostat, a potent inhibitor of HDAC proteins has demonstrated anti tumor activity in animal models and in humans. The purpose of this study was to examine the pharmacokinetic and pharmacodynamic properties of warfarin in combination with belinostat and to evaluate the safety profile of belinostat with concomitant warfarin.

Methods: Eligible patients enrolled on the study received 5 mg PO warfarin 14 days prior to administration of belinostat. Belinostat was administered as an iv infusion, 1000mg/m2 over 30 minutes for 5 consecutive days every 21 days. On day 3, cycle 1 of belinostat treatment, a second dose of 5mg PO warfarin was administered 2 hours prior to belinostat. Pharmacokinetic blood samples were obtained during cycle 1 of the study to measure warfarin and belinostat metabolism. Toxicities were monitored regularly throughout treatment and response was monitored according to standard of care guidelines.

Results: 18 patients, with solid tumors or hematologic malignancies, treated with belinostat and warfarin were included in this analysis. Median age was 55 years (31-77). 11 (61%) patients were male and 7(39%) patients were female. The most common Grade 1 or 2, toxicities observed during the study were anemia (78%), fatigue (72%) and nausea (61%). The most frequent Grade 3 or 4 toxicities were nausea (11%) and hyperuricemia (11%). No Grade 3 or 4 thrombocytopenia or neutropenia were reported. No treatment related Grade 5 toxicities were reported. During cycle 1 no patient experienced treatment delays or discontinued study as result of treatment related toxicity.

Conclusion: Belinostat was generally well tolerated in patients with solid tumors or hematologic malignancies with the major toxicity being anemia, fatigue or nausea. Pharmacokinetic results will be presented at the conference.

Citation Format: Neeraj Agarwal, Mark L. Wade, Julia Batten, Cynthia Davidson, Show-Li Sun, Sunil Sharma. Phase I study of histone deacetylase inhibitor belinostat in combination with warfarin in patients with solid tumors or hematological malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1172. doi:10.1158/1538-7445.AM2013-1172
Abstract 1279: Serum biochemistry and microarray analysis of liver and colonic mucosa in LETO and diabetic OLETF rats treated with 20% caloric restriction and high-fat diet.
Authors: Izumi Keisuke , Takasu Chie , Takahashi Tetsuyuki , Uehara Hisanori
Published: August 2014

We showed recently that diabetic and mildly obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats were more susceptible to azoxymethane-induced carcinogenesis than control Long-Evans Tokushima Otsuka (LETO) rats, and carcinogenesis was inhibited by 20% caloric restriction (CR) and enhanced by high-fat diet (HFD, control diet with 10% safflower oil). To elucidate the mechanism of cancer susceptibility, we performed serum biochemical analysis and microarray analysis of the liver and colonic mucosa. Male LETO and OLETF rats were given control diet, 20% CR diet or HFD for 5 weeks, and euthanized at 24 weeks of age (n=5, respectively). In both strains, blood glucose, serum triglyceride, total cholesterol, insulin and leptin levels decreased by 20% CR, and increased by HFD. Steatosis of hepatocytes in OLETF rats was ameliorated by 20% CR and enhanced by HFD. Microarray analysis of the liver showed that 20% CR up-regulated metallothionein 1A gene, and down-regulated heat shock 105kDa/110kDa protein 1 (Hsph1) and heat shock 70kDa protein 1B (Hspa1b) genes in both strains. There were no histological differences in the colonic mucosa in both strains and the effect of the dietary difference was not found. Microarray analysis of the colonic mucosa showed that 20% CR down-regulated Hsph1 and Hspa1b genes, and HFD up-regulated fatty acid binding protein 2 in both strains. These data suggest that hyperinsulinemia, hyperlipidemia and oxidative stress by overnutrition exert significant role in cancer susceptibility in OLETF rats.

Citation Format: Keisuke Izumi, Chie Takasu, Tetsuyuki Takahashi, Hisanori Uehara. Serum biochemistry and microarray analysis of liver and colonic mucosa in LETO and diabetic OLETF rats treated with 20% caloric restriction and high-fat diet. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1279. doi:10.1158/1538-7445.AM2013-1279
Abstract 1429: Role of monocyte chemotactic protein-1 (MCP-1) in the tumor microenvironment.
Authors: Kucheryavykh Lilia Y. , Rivera-Pagán Aixa F. , Rolón-Reyes Kimberleve , Skatchkov Serguey N. , Eaton Misty J.
Published: August 2014

Glioblastomas, the most malignant form of gliomas, harbor multiple cell types. In particular, microglial cells can contribute up to 30% of a brain tumor mass, and can promote glioma cell growth and dispersal. The purpose of the present study was to test the hypothesis that glioma cells recruit nearby microglia through an MCP-1-mediated mechanism and enhance their production of MCP-1 in the tumor microenvironment. We evaluated the role of MCP-1 on glioma cell proliferation and invasion. Consistent with previous studies in rat models (Platten et al., 2003), we found that U-87 and A-172 human glioma cells promote microglial migration towards the glioma environment using a modified Boyden chamber migration assay. Furthermore, this effect was decreased after immunoneutralization of MCP-1 released from glioma cells. Additionally, using an antibody array we found that U-87 or A-172 glioma cell conditioned medium stimulates a 7-10 fold increase of MCP-1 release by microglia by 24 hours. We next analyzed the consequences of elevated MCP-1 on the invasiveness and proliferation of glioma cells. Using standard invasion assays with or without microglia in a lower compartment, we demonstrated that microglia significantly increased invasion of glioma cells, but this effect was not blocked by MCP-1 immunoneutralization. This suggests that the stimulatory effect of microglia on glioma invasion was due to some other factor such as release of matrix metalloproteinases (MMPs). In contrast, microglia increased U-87 and A-172 glioma cell proliferation within 72 hours and MCP-1 immunoneutralization reversed this proliferative effect of microglia in both cell lines.

Based on these results, we propose that glioma cells release low levels of MCP-1 to recruit nearby microglia and enhance the production of MCP-1. This increased secretion of MCP-1 from microglial cells recruits more microglial cells into the tumor and stimulate glioma progression. A variety of substances released by microglia in tumor microenvironment, including cytokines, growth factors, proteases and extracellular matrix components could lead to increased proliferation and invasiveness of glioma cells.

Supported by G11 HD052352, G12 RR03035, 8G12MD007583-27, U54 NS039408 and by the UCC Pilot Project Program

Citation Format: Lilia Y. Kucheryavykh, Aixa F. Rivera-Pagán, Kimberleve Rolón-Reyes, Serguey N. Skatchkov, Misty J. Eaton. Role of monocyte chemotactic protein-1 (MCP-1) in the tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1429. doi:10.1158/1538-7445.AM2013-1429
Abstract 1459: Detection of EpCAM negative circulating tumor cells in CellSearch waste.
Authors: van Dalum Guus , Lenferink Aufried T.M. , Terstappen Leon W.M.M.
Published: August 2014

Introduction: Circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. The CellSearch system uses immunomagnetic enrichment targeting the EpCAM antigen. A frequently raised question is what the frequency and significance is of CTC that do not express EpCAM within the CTC of the individual patient and between different patients. To investigate this, a device was constructed that collects the blood discarded by the CellSearch system and passes the blood through micro sieves with 5um pores to enrich for the larger CTC.

Methods: A sample collection device was attached to the waste line of the CellTracks Autoprep (AP). The blood discarded after immunomagnetic separation was detected in the waste line and collected into a separate 50ml conical tube for each patient sample. After collection, the blood was filtered through microfabricated silicon nitride filters with pore diameters of 5 micrometer (Aquamarijn, Zutphen, The Netherlands) with a pressure below 40mbar. To evaluate recovery on the filter the COLO320(median size 11μm), SKBR3(16μm) and T24(16μm) cell lines where used. To evaluate recovery from the AP waste the SKBR3(445000 EpCAM antigens) and T24(2167 EpCAM antigens) cell lines where used. 300 cells where spiked in 7.5ml of blood collected in CellSave tubes from healthy volunteers. All samples were processed the day after collection.

Results: The average recovery on filter from whole blood is 29% for COLO320 and ≥80% for SKBR3 and T24. The average recovery of the SKBR3 cell line with the CellSearch system is 91% and 5% are recovered from the AP waste using filtration. In contrast, the average recovery of T24 cell line with the CellSearch system is only 30% but 51% are recovered from the AP waste after filtration. The carryover between spiked and unspiked samples collected from the AP waste was <1.3% of the spiked cells for unspiked samples with a mean of 0.9%.

Conclusions: We present a method and device that enables the identification and characterization of CTC not detected by the CellSearch system which allows a systematic evaluation of the clinical relevance of these CTC.

Citation Format: Guus van Dalum, Aufried T.M. Lenferink, Leon W.M.M. Terstappen. Detection of EpCAM negative circulating tumor cells in CellSearch waste. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1459. doi:10.1158/1538-7445.AM2013-1459