bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2432: AMF/PGI modulates EGF-induced invasion of breast cancer cell line via positive feedback loop
Authors: Kho Dhonghyo , Raz Avraham
Published: June 2012

Phosphoglucose isomerase (PGI/AMF) is well established to promote cell migration as an autocrine motility factor. It remains to be established which microenvironmental signals enhance the secretion of AMF from tumor cells. Herein, we found that epithelial growth factor (EGF) enhances the secretion of AMF in the breast cancer cell line, T47D. Immunoprecipitation study shows that Caisen kinase 2α (CK2α) increases the phosphorylation of intracellular AMF. Therefore, increasing secretion of AMF and the extracellular AMF enhances the downstream cascade of EGF such as Phospho-ERK and β-catenin stabilization. In the transwell motility study, EGF-induced invasion increases synergistically with purified AMF. To confirm this, we demonstrate that the knockdown of AMF by siRNA treatment suppresses EGF-induced invasiveness. These findings imply that AMF may modulate the EGF-induced invasion in the tumor microenvironment.

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 2432. doi:10.1158/1538-7445.AM2011-2432
Abstract 2434: CXCR3-mediated cellular and molecular events involved in breast cancer metastasis
Authors: Li Yanchun , Reader Jocelyn , Ma Xinrong , Kundu Namita , Kao Joseph P. , Zhan Steven , Fulton Amy M.
Published: June 2012

We previously detected chemokine receptor CXCR3 in malignant epithelium of early stage breast cancers by immunohistochemistry and the expression levels were positively correlated with significantly poorer survival. We also showed that CXCR3 contributes to metastatic potential in a murine model of metastatic breast cancer. The goal of the present study is to determine the cellular and molecular mechanisms underlying CXCR3-mediated breast cancer metastasis. Treatment of the human breast cancer cell line MDA-MB-231 with the CXCR3 ligand CXCL10 induced calcium mobilization and Rac activation; both responses are essential for tumor cell chemotaxis. Not surprisingly, CXCL10 enhanced migration and invasion of MDA-MB-231 cells. We also showed that CXCL10 promoted anchorage independent growth of both human malignant mammary epithelial cells (MDA-MB-231 and MDA-MB-468) and murine malignant mammary epithelial cell line 66.1 in an ERK- dependent manner. In tumor cells, ERK1/2 is robustly activated after CXCR3 ligand stimulation, whereas the same pathway is inhibited or not changed in normal cells. In addition, CXCR3 activation provided some degree of anoikis resistance to 66.1 cells. There are two isoforms of CXCR3 in human cells. The “classical” CXCR3 receptor (CXCR3-A) mediates proliferation and migration of various cell lines, while CXCR3-B, a splice variant of CXCR3-A, has opposing functions. We determined the expression pattern of these two CXCR3 isoforms in a panel of human breast cancer cell lines by real-time quantitative PCR and showed that the CXCR3-B/CXCR3-A mRNA expression ratio is decreased (0.16-0.63) compared with normal epithelial cell line MCF-10A (2.16). Taken together, these data indicate that multiple molecular and cellular mechanisms may contribute to CXCR3-mediated breast cancer metastasis. Due to the distinct functions of CXCR3-A and CXCR3-B, the relative amounts of CXCR3-A and CXCR3-B may dictate cell behavior. Further studies will investigate how the balance between CXCR3-A and CXCR3-B regulates the metastatic potential of breast cancer cells and allow us to optimize therapeutic strategies targeting CXCR3.

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 2434. doi:10.1158/1538-7445.AM2011-2434
Abstract 2435: Chromogranin A and its N-terminal fragment affect the metastatic potential of circulating tumor cells in mice
Authors: Dondossola Eleonora , Crippa Luca , Colombo Barbara , Ferrero Elisabetta , Corti Angelo
Published: June 2012

Chromogranin A (CgA) is an acidic glycoprotein present within secretory vesicles of many normal and neoplastic endocrine and neuroendocrine cells. CgA is abnormally released in circulation in patients with neuroendocrine tumors and non neuroendocrine tumors, as in a subpopulation of patients with non small cell lung cancer (NSCLC), with important prognostic implications. We have previously demonstrated that CgA can preserve the barrier function of the vascular endothelium. Since hematogenous metastasis requires the seeding of malignant cancer cells in circulation, followed by cell extravasation and colonization of distant organs, we investigated the role of CgA on tumor cell extravasation and lung colony formation. We show that normal levels of endogenous CgA can reduce the number of lung colonies from mammary TS/A adenocarcinoma cells injected into the venous circulation of mice. Administration of omeprazole, a drug commonly used in the treatment of acid-peptic disorders, increased the blood levels of CgA and further inhibited the development of lung colonies in a CgA-dependent manner. Furthermore, administration of recombinant CgA or its N-terminal fragment (called vasostatin-1, VS-1) reduced the metastatic potential of circulating mammary TS/A adenocarcinoma and B16-F10 melanoma cells. Studies on the mechanism of action showed that CgA and VS-1 can inhibit the transendothelial migration of cancer cells, by preserving the VE-cadherin-dependent endothelial barrier function. These findings suggest that endogenous CgA can play a role as a modulator of the metastatic potential of circulating tumor cells.

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 2435. doi:10.1158/1538-7445.AM2011-2435
Abstract 2442: XIAP mediates cancer cell motility via RhoGDI-dependent regulation of cytoskeleton
Authors: Yu Yonghui , Liu Jinyi , Zhang Dongyun , Luo Wenjing , Yu Jianxiu , Li Jingxia , Zhang Xinhai , Wu Xue-Ru , Acosta Germán R. , Huang Chuanshu
Published: June 2012

XIAP overexpression has been found to be associated with malignant cancer progression and aggression in individuals with many types of cancers. However, the molecular basis of XIAP in the regulation of cancer cell biological behavior remains largely unknown. In the current study, we found that the deficiency of XIAP expression in human cancer cells by approach of either knockout or knockdown led to a marked reduction of β-Actin polymerization and cytoskeleton formation. Consistently, cell migration and invasion were also decreased in XIAP-deficient cells in comparison to those in the parent wild type cells. Subsequent studies demonstrated that the regulation of cell motility by XIAP depended on its interaction with RhoGDI via XIAP's RING domain. Furthermore, XIAP was found to negatively regulate RhoGDI SUMOylation, which might affect its activity in controlling cell motility. Collectively, our studies provide novel insights into the molecular mechanisms by which XIAP regulates cancer invasion, and offer further theoretical basis for setting XIAP as a potential prognostic marker and specific target for treatment of cancers with metastatic property.

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 2442. doi:10.1158/1538-7445.AM2011-2442
Abstract 2445: CD24+ liver tumor-initiating cells drives self-renewal and tumor initiation through Stat3-mediated Nanog regulation
Authors: Lee Kin Wah , Castilho Antonia , Cheung Vincent Chi-Ho , Tang Kwan Ho , Ma Stephanie , Ng Irene Oi-Lin
Published: June 2012

Tumor-initiating cells (TICs) are a subpopulation of chemoresistant tumor cells that have been shown clinically to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways are therefore priorities for the development of novel therapeutic paradigms. We established chemoresistant hepatocellular carcinoma (HCC) xenograft tumors in immunocompromised mice in which an enriched T-IC population was capable of tumor initiation and self-renewal. Using this model, we found CD24 to be up-regulated in residual chemoresistant tumor tissue when compared with bulk tumor upon cisplatin treatment. The determine whether CD24 is a candidate TIC marker for HCC, cell sorting approach was employed to separate CD24-/CD24+ populations derived from HCC cell lines and clinical samples. CD24+ HCC cells were found to be critical for the maintenance of tumor growth, self-renewal, differentiation, chemoresistance and metastasis of tumors. Using quantitative PCR, CD24 was over-expressed in HCCs when compared with their non-tumor counterparts, and CD24 expression was significantly correlated with poor patients’ survival.

Using a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives tumor initiation and self-renewal. Notably, whether by cell sorting or gene-knockdown approach, it was shown by quantitative PCR that the CD24-enriched population consistently overexpressed Nanog. Upon transfection of Nanog cDNA into CD24-knockdown cells, self-renewal and tumor formation were functionally recovered, suggesting Nanog as the downstream effecter of CD24. By Ingenuity Pathway analysis, ‘acute phase response signaling’ was found to be the most significantly altered upon CD24 knockdown in which phosphorylation of Stat3 at the Tyrosine705 residue is critical. Using Stat3 inhibitor (S3I-201), we found that Nanog promoter and its protein were down-regulated in dose-dependent manner in CD24+ HCC cells. CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through Stat3-mediated Nanog regulation.

In conclusion, we identified CD24+ HCC cells within bulky tumor, and they functioned to initiate tumor growth and self-renewal through Stat3-mediated Nanog up-regulation. The identification of novel CD24 signaling pathways provides an attractive therapeutic strategy against this deadly disease.

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 2445. doi:10.1158/1538-7445.AM2011-2445
Abstract 2448: Inhibiting the DLL4/Notch pathway delays tumor recurrence and decreases tumorigenicity in pancreatic cancer
Authors: Fischer Marcus , Yen Wan-Ching , Beviglia Lucia , Yeung Pete , Lewicki John , Gurney Austin , Hoey Timothy
Published: June 2012

It is becoming increasingly evident that cancer stem cells drive tumor growth and progression and are preferentially resistant to chemotherapy. Previously, we demonstrated that interfering with the Delta-like 4 ligand (DLL4)/Notch signaling by anti-DLL4 antibodies decreased tumor growth in a pancreatic xenograft model, delayed tumor recurrence following chemotherapy, and reduced the pancreatic cancer stem cell (CSC) fraction.

In this study, we evaluated the response of seven pancreatic xenograft tumors to anti-DLL4 antibodies as a single agent and when combined with gemcitabine. Utilizing an in vivo limiting dilution assay, we tested the ability of cells from previously treated xenografts to re-grow tumors after serial transplantation to quantify CSCs. We discovered that anti-DLL4 was efficacious as a single agent in all seven tumor models. In all seven models, when anti-DLL4 was combined with gemcitabine, the therapy provided significant efficacy over that of gemcitabine alone. This combination therapy resulted in tumor regression in three of these models. The antibody therapy reduced the CSC fraction, while gemcitabine treatment alone enriched for the CSC. The chemotherapy/antibody combination further reduced the CSC fraction compared to anti-DLL4 alone.

To analyze the mechanism of action of inhibiting DLL4-Notch signaling in tumor cells and in the host vasculature, we tested a human specific DLL4 mAb and a murine specific DLL4 mAb as single agents. Anti-human DLL4 reduced the tumorigenicity of pancreatic tumor cells, while anti-murine DLL4 had no effect on tumor initiating cell frequency. In a tumor recurrence model, the human specific DLL4 mAb delayed tumor recurrence following the termination of gemcitabine while the murine specific DLL4 mAb decreased the growth rate of recurrent tumors. The combination of human and murine targeting DLL4 mAbs produced an additive effect in reducing tumor recurrence. These data support the rationale for targeting the DLL4/Notch pathway in pancreatic cancer and provide evidence of the therapeutic utility of targeting pathways, such as Notch, required for cancer stem cell self renewal.

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 2448. doi:10.1158/1538-7445.AM2011-2448
Abstract 2469: ATM inhibitor 55933 abolishes the radiation resistance of CD44+/CD24−or low subsets of breast cancer cells
Authors: Yin Hong , Glass Jonathan
Published: June 2012

CD44+/CD24− subsets of breast cancer cells are enriched in cancer stem-like cells (CSC) and are radiation resistant. We isolated ESA+/CD44+/CD24− or low subsets by flow cytometry from MCF7, MDA-MB231, and HCC1937 breast cancer cell lines and primary cultures of breast cancer cells from two patients. The isolated CD44+/CD24−orlow subsets showed a 50 to 70% increase of sphere formation by in suspension culture in serum-free medium compared to CD44−/CD24high subsets. CD44+/CD24−or low subsets of the cell lines and primary cultures showed a significantly increased radiation resistance compared to the CD44−/CD24high cell line subsets or the unsorted primary cells with > 40% increased survival at 2Gy. Post-radiation survival showed that the radiation resistance was dependent on the CD44+/CD24 −or low cell phenotype. The correlation of CD44/CD24 status with radiation resistance was demonstrated by culture of the subsets. After 2 weeks of culture the following transitions occurred: 1) CD44+/CD24−or low cells changed to CD44−/CD24high cells (MCF-7 and HCC1937) or to CD44+/CD24+ (MDA-MB-231); and 2) CD44−/CD24 high (MCF-7 and HCC1937) and CD44+/CD24+ (MDA-MB-231) cells became CD44+/CD24−or low cells. Cells that lost the CD44+/CD24 −or low phenotype were no longer radiation resistant. The mechanisms of increased radiation resistance in CD44+/CD24−orlow subsets was not a function of non-homologous end joining (NHEJ) as there was no in vivo and in vitro change in NHEJ activity in the resistant cells nor altered expression of the NHEJ related proteins Ku 80, Ku70, PARP-1, and DNA-PK. However, there were differences ATM activity between CD44+/CD24−or low and the CD44−/CD24 high (MCF-7 and primary breast cancer) or CD44+/CD24+ (MDA-MB-231) subsets. Depending on the cell type and radiation dose, ATM phsophorylation increased by 2-5 fold in radiated CD44+/CD24−or low subsets of breast cancer cell lines and primary cultured breast cancer cells compared to the control subsets. The ATM inhibitor 55933 in a dose-dependent manner increased radiation sensitivity of all subsets including significantly abolishing the radiation resistance of CD44+/CD24−or low subsets of cells. After 2Gy of radiation, the survival fractions of cells treated with10μM of 55933 were 8-10 fold lower than that of untreated cells and were now similar between CD44+/CD24−or low subsets and control subsets for the cell lines and primary cultures. In conclusion, ATM is a candidate target for eliminating the radiation resistance of CSC's of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2011-2469
Abstract 2479: Detecting Abelson tyrosine kinase activation by ATM and DNA-PK after exposure to ionizing radiation using a peptide biosensor
Authors: Tang Jiabin , Wang Jean Y. , Parker Laurie
Published: June 2012

Abelson (Abl) kinase is a non-receptor tyrosine kinase that is involved in many cellular processes, including survival, differentiation and apoptosis. Abl is activated in the response to DNA damage, and is involved in the decision between DNA repair versus apoptosis. It is widely accepted that Abl is regulated in response to DNA double-strand breaks (DSBs) by ataxia telangiectasia mutated protein (ATM) via phosphorylation of Abl at S465. However, other aspects of this complex signaling response have been unclear, including the role of other kinases such as DNA-dependent protein kinase (DNA-PK), because of the difficulties of detecting subtle changes in Abl activation through endogenous substrates. Here, we show Abl is activated in a mutant cell line that cannot be phosphorylated by ATM at S465 (Abl-S465A-EGFP) after exposure to ionizing radiation (IR) using a peptide biosensor we developed for Abl kinase. This biosensor contains the following functional modules: a highly specific Abl kinase substrate, an Abl SH3 binding ligand, a photocleavable linker, a biotin tag and a cell penetrating peptide derived from Hiv-TAT to aid intracellular delivery of cargo across the plasma membrane. Using a combination of chemical biology approaches, Western blot for the phosphorylation of the biosensor peptide and high-resolution MS-based phosphoproteomics, we identified a potential role for DNA-PK in activating Abl kinase after IR, independent of ATM and S465. This information about the importance of DNA-PK in this signaling pathway may lead to a better understanding of c-Abl's function in the response to IR, and could be useful to develop strategies to combat radioresistance or improve radiosensitivity in tumors.

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 2479. doi:10.1158/1538-7445.AM2011-2479
Abstract 2480: Low dose radiation- and thiol-induced effects on micronuclei formation following fractionated irradiation
Authors: Murley Jeffrey S. , Miller Richard C. , Baker Kenneth L. , Murley Tyler S. , Miller Kristin L. , Sullivan Joseph T. , Wardak Hamid , Grdina David J.
Published: June 2012

Exposure of cells to low doses of ionizing radiation (≤ 10 cGy) or amifostine induces pro-survival pathways that result in protection against the damaging effects of high dose ionizing radiation exposure. We have identified one such signaling pathway whose molecular mechanism involves the elevation of active manganese superoxide dismutase (SOD2) levels in the cell. SOD2 is a mitochondrial matrix protein that serves as the primary mitochondrial defense against superoxide formation. Treatment of RKO36 human colon carcinoma cells with 40 μM WR1065 or 10 cGy x-rays causes a significant increase in SOD2 activity that peaks 24 h after exposure. This increase in SOD2 activity is completely eliminated in cells subsequently exposed to 2 Gy. Following the dismutation of O2−, SOD2 is rapidly converted back into active enzyme in the case where enzyme and substrate concentrations are approximately equal. However, when substrate concentration is much greater than enzyme concentration, as is the case following exposure to 2 Gy radiation, the conversion from inactive to active enzyme occurs with much slower kinetics, taking minutes rather than seconds to occur. The protective effect of the thiol- and radiation-induced elevation in active SOD2 was examined using the endpoint of micronuclei formation as a measure of chromosomal damage. Exposure of RKO36 cells to 4 Gy results in a significant increase in the mean number of micronuclei observed. The frequency of micronuclei formation is significantly reduced when cells are treated with 40 μM WR1065 or exposed to 10 cGy 24 h prior to being irradiated with 4 Gy. No protective response is observed in cells exposed to 40 μM WR1065 30 min, 1 h or 3 h prior to two 2 Gy fractionated radiation doses separated by 24 h, or in cells exposed to 10 cGy 30 min, 3 h or 6 h before the first of two 2 Gy fractions. Treatment of cells with 40 μM WR1065 30 min, 1 h or 3 h after the first of the two 2 Gy fractions, or with 10 cGy 30 min, 3 h or 6 h after the first 2 Gy fraction also did not protect cells against the formation of micronuclei relative to cells exposed to the 2 Gy fractions alone. This work was supported by NIH/NCI grant R01 CA132998 and DOE Low Dose Program/Project Grant DE-SC0001271 (D.J.G).

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 2480. doi:10.1158/1538-7445.AM2011-2480
Abstract 2526: Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity
Authors: Ganapathi Mahrukh K. , Grozav Adrian G. , Willard Belinda B. , Kozuki Toshiyuki , Chikamori Kenichi , Micluta Marius A. , Petrescu Andrei-Jose , Kinter Michael , Ganapathi Ram N.
Published: June 2012

Topoisomerase (topo) II catalyzes topological changes in DNA. Although, both human isozymes, topo IIα and β, are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin we detected four +80-Da modified sites, tyrosine 656, serine 1395, threonine 1426 and serine 1545. Phosphorylation at serine 1395, threonine 1426 and serine 1545 was established based on neutral loss of H3PO4 (−98 Da) in the collision-induced dissociation (CID) spectra and on differences in 2D-phosphopeptide maps of 32P-labeled wild type (WT) and serine1395alanine or threonine1426alanine/serine1545alanine mutant topo IIβ. However, phosphorylation at tyrosine 656 could not be verified by 2D-phosphopeptide mapping of 32P-labeled WT and tyrosine656phenylalanine mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyrosine 656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions (characteristic of bromination) in CID spectra of two peptides containing tyrosine 656 and tyrosine 711, confirming bromination. Interestingly, mutation of only tyrosine 656, but not serine 1395, threonine 1426 or serine 1545, decreased topo IIβ activity suggesting a functional role for tyrosine 656. Homology modeling of the region of topo IIβ (amino acids 449-1108) containing these two reactive tyrosine residues revealed that tyrosine 711 is buried, whereas tyrosine 656 is highly exposed on the surface. This region is within, or very close to the dimerization interface, which suggests that these amino acids may be critical for topo IIβ dimer formation. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.

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 2526. doi:10.1158/1538-7445.AM2011-2526
Abstract 2542: Preclinical studies of novel phosphonium salts with broad-spectrum activity
Authors: Millard Melissa , Pathania Divya , Shabaik Yumna , Taheri Laleh , Deng Jinxia , Neamati Nouri
Published: June 2012

Proliferation under selective pressures exerted by an unstable microenvironment, requires tumor cells employ adaptive mechanisms that confer growth advantage. Adaptation to adverse conditions results in cellular phenotypes that typify neoplastic transformation and offer unique opportunities for selective targeting of cancer cells. Agents that target tumor cell mitochondria with high selectivity hold clinical significance due to the adaptive, modulatory and essential role of this organelle in cancer cell energy production, metabolism and apoptosis. To this end, we have identified a series of novel, mitochondriotropic phosphonium salts, (TP compounds), that have shown broad-spectrum anti-cancer and anti-angiogenic activity in preclinical evaluation. A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 and numerous close analogues as having IC50 concentrations in the sub-micromolar range. TP treatment induced cell cycle arrest, lowered oxygen consumption, and increased mitochondrial superoxide production. Administered as single agents in a mouse model of human breast cancer, TP compounds significantly decreased tumor growth with no observed toxicities. Protein microarray data demonstrated significant down-regulation of integrin and growth factor mediated signaling pathways governing key processes including cancer cell survival, proliferation and tumor angiogenesis. At low micromolar concentrations, TP compounds prevented integrin-mediated cell adhesion to fibronectin and vitronectin coated substrates and tumor cell haptotaxsis on vitronectin coated boyden chambers. Similar concentrations of TP compound also prevented growth factor induced endothelial cell tube formation in 3-D culture using basement membrane extracts. Taken together, these results suggest that as mitochondria-targeted agents, TP compounds act to inhibit tumor cell proliferation and angiogenic capacity.

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 2542. doi:10.1158/1538-7445.AM2011-2542
Abstract 2547: NCI-60 combination screening matrix of approved anticancer drugs
Authors: Holbeck Susan L. , Collins Jerry M. , Doroshow James H.
Published: June 2012

Anticancer drugs are rarely curative as single agents, thus most treatment regimens utilize a combination of agents. Selection of rational combinations based upon presumed mechanisms of action is an active area of research. Nonetheless, treatment regimens may have built up over time by addition of new agents to existing standards of care. In order to establish a framework for extending our understanding combination therapy, we have initiated an additional approach: the systematic testing of all pairwise combinations of FDA-approved anticancer drugs in the NCI-60 panel of human tumor cell lines. About 100 small molecule drugs are approved for cancer treatment worldwide – testing all 5000 pairwise combinations in each of 60 cell lines will require 300,000 experiments. Combinations are tested at 3 or more concentrations of each agent, and single agents are tested on the same plates, requiring ∼ 5 million wells. To date, about 100 pairs of drugs have been tested in the NCI-60 in a pilot phase – the high-throughput phase has just begun. Some combinations of drugs, such as bortezomib combined with cladribine, show better than single agent activity in nearly all cell lines tested. Other combinations, such as doxorubicin plus lapatinib, show more restricted benefit, with some cell lines showing enhanced growth inhibition or cell kill, while in other cell lines the combination is antagonistic or identical to the more active single agent. The NCI-60 panel of cell lines has been extensively molecularly characterized, with publicly available data including gene mutation, DNA copy number, DNA methylation, and expression of mRNA, protein and microRNA. The patterns of cell line sensitivity for a particular drug combination can be used to probe these molecular characterization data, generating hypotheses about potential predictive markers and/or mechanisms of action. Probing the doxorubicin plus lapatinib patterns reveals a strong correlation with MDR activity, with high MDR expressing lines showing enhanced combination activity. The most promising combinations identified from NCI-60 testing are being tested in vivo to determine the therapeutic index. Already 2 combinations have demonstrated promising xenograft activity. The combination screening data, along with tools to analyze the data, will be made available to the public through the NCI web site.

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 2547. doi:10.1158/1538-7445.AM2011-2547
Abstract 2548: Chemosensitization of cancer cells by KU-0060648: A dual inhibitor of DNA-PK and PI-3K
Authors: Batey Michael A. , Munck Joanne M. , Zhao Yan , Cano Celine , Tavecchio Michele , Bardos Julia , Griffin Roger J. , Jenkins Helen , Menear Keith , Smith Graeme , Thommes Pia , Newell David R. , Martin Niall , Curtin Nicola J.
Published: June 2012

DNA double strand breaks (DSBs) are the most cytotoxic lesions induced by topoisomerase II poisons. Enzyme-mediated repair of DNA DSBs is a major mechanism of resistance to DNA DSB-inducing agents. Non-homologous end joining (NHEJ) is the major pathway for the repair of DSBs and requires DNA-PK activity. DNA-PK has close structural similarities to PI-3K, which promotes cell survival and proliferation and is up-regulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. We investigated the therapeutic utility of KU-0060648, in models of human cancer.

KU-0060648 was investigated in a panel of human breast and colon cancer cells, it inhibited cellular DNA-PK auto-phosphorylation with IC50 values of 0.019 µM (MCF7 cells) and 0.17 µM (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC50 values of 0.039 µM (MCF7 cells) and >10 µM (SW620 cells). 5-day exposure to 1 µM KU-0060648 inhibited cell proliferation by > 95% in MCF7 cells, but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitisation were maintained within the tumour for at least 4 hours at non-toxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumour growth delay in both SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels.

The proof of principle in vitro and in vivo chemosensitisation with KU-0060648 justifies further evaluation and characterisation of dual DNA-PK and PI-3K inhibitors.

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 2548. doi:10.1158/1538-7445.AM2011-2548
Abstract 2551: The Nampt inhibitor MPC-9528 synergizes with DNA damaging agents
Authors: Terry-Lorenzo Ryan T. , Douce Thomas B. , Austin Harry A. , Boniface J. Jay , Cimbora Daniel M. , Fleischer Tracey C. , Carlson Robert O.
Published: June 2012

Background: MPC-9528 reduces cellular NAD levels and causes cell death, by blocking the NAD salvage pathway through inhibition of nicotinamide phosphoribosyltransferase (Nampt). Many DNA damaging agents also reduce cellular NAD levels, by activating the NAD consuming enzyme poly(ADP-ribose) polymerase (Parp). We hypothesized that the combination of a Nampt inhibitor and a DNA damaging agent would synergize in killing cancer cells, due to a combined effect on NAD levels through two independent mechanisms.

Methods: Cellular NAD was measured using a coupled enzymatic assay. Drug combination experiments were performed in HCT116 colon carcinoma cells, using measurement of ATP levels as a cell viability endpoint. Synergy, antagonism, or additivity was assessed using the MacSynergy II program.

Results: In HCT116 cells, saturating doses of MPC-9528 induced depletion of NAD with a half-life of 5 hours and a decrease in ATP that was delayed approximately 14 hours relative to NAD. Lower, sublethal concentrations of MPC-9528 induced partial NAD depletion without a concomitant ATP loss. At these sublethal concentrations, MPC-9528 was found to synergize with the DNA alkylating agents temozolomide and streptozotocin, which are known to activate Parp. Additionally, MPC-9528 was found to synergize with two structurally different thymidylate synthase inhibitors, 5-fluorouracil (5-FU) and raltitrexed, neither of which have been reported to activate Parp. Individually, 5-FU and raltitrexed each caused NAD depletion in HCT116 cells, which was enhanced by combination with MPC-9528. Furthermore, both 5-FU- and raltitrexed-mediated NAD depletion and synergy with MPC-9528 were completely blocked by the Parp inhibitor olaparib.

Conclusions: Parp activation induced by the alkylating agents temozolomide and streptozotocin, or by the thymidylate synthase inhibitors 5-FU and raltitrexed, is the basis for tumoricidal synergy with the Nampt inhibitor MPC-9528. This synergy is a direct consequence of the NAD depletion resulting from Parp activation coupled with the inhibition of NAD synthesis due to Nampt inhibition. These results provide a basis for clinical combination of MPC-9528 with the agents studied here or with related agents that induce Parp activation.

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 2551. doi:10.1158/1538-7445.AM2011-2551
Abstract 2559: Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma
Authors: Saunders Nicholas A. , Erlich Rafael , Kherrouche Zoulika , Hazar-Rethinam Mehlika , de Long Lilia Merida , Guminski Alexander
Published: June 2012

In this study we have examined the potential value of a series of clinically relevant PI3K-mTOR inhibitors alone, or in combination with histone deacetylase inhibitors, in a model of head and neck squamous cell carcinoma (HNSCC). We present data relating to in vitro and in vivo pharmacological effects and efficacy for both these classes of drugs.

HNSCC cell lines, human keratinocyte and HNSCC xenograft models were treated with histone deacetylase inhibitors (HDACIs) and new generation PI3K and dual PI3K-mTOR inhibitors either alone or in combination. Cell and tumour tissue viability and proliferation were determined in vitro and in vivo following treatments. Effect upon activation status of signalling pathways was also investigated.

We report that PI3K, AKT, and dual PI3K-mTOR inhibitors (LY294002, Wortmannin, AKT VIII, BEZ235, BKM120 and BGT226) caused a marked in vitro enhancement of cytotoxicity induced by HDACIs (vorinostat, depsipeptide, valproic acid and LBH589) in HNSCC cancer cells (SCC25, Cal27, Detroit 562). This effect correlates with intense and duration of AKT inhibition, reactive oxygen species (ROS) accumulation, and is attenuated by expression of constitutively active AKT. LBH589, BEZ235, BGT226 and BKM120 inhibited tumour growth alone and in combination in a xenograft model of HNSCC. However, we saw no evidence of improved efficacy with an HDACI / PI3KI combination. Importantly, we observed intratumoural HDAC inhibition and PI3K inhibition as assessed by histone H3 acetylation status and phospho-AKT staining respectively. Finally, further studies indicated that the PI3KI/mTOR inhibitor, BGT226, could significantly enhance cisplatin or taxane-induced cytotoxicity in HNSCC cell lines in vitro.This study indicates, for the first time, that new generation clinically relevant PI3K and dual PI3K-mTOR inhibitors possess antitumour effect against SCC in vivo. These findings provide a rational basis for pursuing clinical trials of PI3KIs in HNSCC.

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 2559. doi:10.1158/1538-7445.AM2011-2559
Abstract 2575: Differentiation of NUT midline carcinoma by epigenomic reprogramming
Authors: Schwartz Brian , Hofer Matthias , Lemieux Madeleine , Bauer Daniel , Cameron Michael , West Nathan , Agoston Elin , Ince Tan , Christie Amanda , Aster Jon , Sallan Stephen , Kung Andrew , Bradner James , French Chris A.
Published: June 2012

NUT midline carcinoma (NMC) is a lethal tumor defined by the presence of BRD-NUT fusion proteins that act by arresting differentiation. Here, we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. We find that overexpression of the twin bromodomains of BRD4 or a portion of NUT that binds the histone acetyltransferase (HAT) p300 induces NMC cells to differentiate, suggesting a model in which BRD4-NUT sequesters HATs in areas of acetylated chromatin. Consistent with this idea, knockdown of BRD4-NUT caused global increases in histone acetylation, whereas enforced expression of BRD4-NUT had the opposite effect. Sequestration of HATs appears to occur in nuclear BRD4-NUT foci, which correspond to specific genomic regions that are rich in acetylated histones but transcriptionally inactive. Of therapeutic interest, treatment of NMC cells with histone deacetylase inhibitors (HDACi) dispersed nuclear foci, restored histone acetylation, induced NMC cells to differentiate in vitro, and had anti-tumor effects in NMC xenograft models. The data suggest that this rational therapeutic approach merits further evaluation in patients with NMC.

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 2575. doi:10.1158/1538-7445.AM2011-2575
Abstract 2600: HMGB1, a potential new target for mesothelioma therapy
Authors: Jube Sandro , Rivera Zeyana , Gaudino Giovanni , Pass Harvey I. , Bianchi Marco E. , Carbone Michele , Yang Haining
Published: June 2012

Malignant mesothelioma (MM) is a very aggressive cancer that causes 2000-3000 deaths annually in the U.S. and is resistant to current therapies. Diagnosis frequently occurs at a late stage of disease progression and median survival from time of diagnosis is about 1 year. Therefore, novel therapeutic strategies are urgently needed. We found previously that High-Mobility Group Box 1 protein (HMGB1), a known pro-inflammatory cytokine, induces tumor necrosis factor-alpha (TNF-α) secretion, potentiates survival of asbestos-exposed primary human mesothelial cells (HM) and enables HM malignant transformation, which leads to the initiation of MM. We show here that HMGB1 is also critical in the maintenance and progression of established MM. We found that HMGB1 is highly expressed in MM tissues and its levels in MM patients’ sera are significantly higher than in those of healthy individuals (P < 0.0001). MM tumor cells over-express HMGB1 in both the nucleus and cytoplasm. Instead, normal HM only express nuclear HMGB1 at significantly lower level than MM cells. The main receptor of HMGB1, receptor for advanced glycation end products (RAGE), is also upregulated in MM cells compared to HM. MM cells release biologically active HMGB1 into extra-cellular space, and the interaction with its receptors induces cell proliferation and chemo-attractant activity on MM cells. Moreover, the interference with HMGB1 activity by using specific HMGB1 inhibitors, such as BoxA and HMGB1 neutralizing monoclonal antibodies, leads to MM cell death. HMGB1 inhibitors impair MM cells migration, invasion, wound healing and also colony formation in soft agar. Our results strongly suggest that HMGB1 can be a novel target for mesothelioma 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 2600. doi:10.1158/1538-7445.AM2011-2600
Abstract 2608: HDAC-C1A: An irreversible HDAC inhibitor with significant anti-tumor activity
Authors: Kaliszczak Maciej , Perumal Meg , Aboagye Eric
Published: June 2012

Histone deacetylase (HDAC) enzymes exert control over gene transcription and cell cycle progression and their inhibition has recently emerged as an efficacious strategy to treat cancer. However, current HDAC inhibitors have been linked to a shared undesirable toxicological profile that prompts the development of new entities. The present study evaluates the in vitro and in vivo PK/PD of a newly developed HDAC inhibitor, HDAC-C1A.

HDAC-C1A inhibited class I, II and sirtuins, with highest affinity for HDAC6 (IC50 = 63 ng/mL), an HDAC subtype thought to be associated with low toxicity; HDAC6 knockout does not lead to embryonic lethality. The drug irreversibly inhibited HDAC from HeLa cell extract; in HCT116 cells inhibition of enzyme activity as assessed by levels of acetyl-histone H3, H4 and acetyl-tubulin was maintained after washout demonstrating an irreversible mechanism. HDAC-C1A treatment was associated with a dose and time dependent increase of histone and non-histone targets that was maintained at 4 hours after washout; acetylation was lost by 4 h with clinically licensed HDAC inhibitor SAHA. HDAC-C1A inhibited the growth of a panel of 7 cancer cell lines with a mean GI50 of 1.6 ± 0.6 µg/mL. The drug was relatively stable after parenteral administration for 4 h. The Cmax and AUC0-4h following i.p. injection (160 mg/kg) were 20.4 µg/mL and 50 µg/mL*h, respectively.

Regarding efficacy, HDAC-C1A treatment was associated with a Tumor Growth Delay (TGD2x) of 5.7 ± 1.4 days and a Tumor Growth Inhibition (TGI) of 78% compared with vehicle when given i.p. at 20 mg/kg b.i.d. in a HCT116 human colon cancer xenograft model. This dose was non-toxic (no reduction in body weight). We also assessed the potential of [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to measure early response to HDAC-C1A treatment in HCT116 xenograft bearing mice. There was a 2-fold decrease in tumor [18F]FLT uptake compared to vehicle treated animals at 48 h post-treatment; the area under the normalized [18F]FLT time versus activity curve was 117 ± 6.9 at before treatment and decreased to 106 ± 5.5 (P = 0.02) at 24 hours and 54 ± 5 (P = 0.0001) at 48 h after initiating treatment.

In summary, HDAC-C1A combines irreversible HDAC inhibition with a favourable pharmacokinetic profile leading to significant anti-tumor activity in the HCT116 tumor model. Early response to the drug was detectable by [18F]FLT-PET. These preclinical data support further development of HDAC-C1A. This work was supported in part by Cancer Research U.K.-Engineering and Physical Sciences Research Council Grant C2536/A10337.

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 2608. doi:10.1158/1538-7445.AM2011-2608
Abstract 2614: Inhibition of class I histone deacetylases attenuates anthracycline induced activation of the ATM pathway
Authors: Thurn Kenneth (Ted) , Thomas Scott , Raha Paromita , Qureshi Ian , Munster Pamela N.
Published: June 2012

Breast cancer is one of the most common cancers to afflict women in the world. Anthracycline-based regimens, including doxorubicin and epirubicin (epidoxorubicin), are common therapies for patients with breast cancer. Doxorubicin and epirubicin are inhibitors of topoisomerase II, which cause double strand DNA breaks. Following anthracycline-induced damage, DNA damage response pathways are activated in order to initiate cell cycle arrest, DNA repair, and/or cell death. The recent introduction of PARP inhibitors highlights the clinical relevance of targeting DNA repair pathways in treating this disease.

Recently, two histone deacetylase (HDAC) inhibitors have been approved by the Federal Drug Administration for the treatment of cutaneous T-cell lymphoma: vorinostat and romidepsin. HDAC inhibitors reduce the repair of DNA, and are being used in combination with DNA damaging chemotherapeutic agents. The mechanisms regulating the HDAC inhibitor-mediated reduction of DNA repair in breast cancer cells are not well established. Understanding these mechanisms will identify key therapeutic targets for reducing DNA repair, define the role of HDACs in DNA repair, and allow for the enrichment of patients most susceptible to HDAC inhibitor-chemotherapy combined treatment.

Previously, our group has demonstrated an increase in DNA decondensation which permits greater access of anthracyclines to DNA after therapeutic inhibition of HDACs. New results demonstrate that phosphorylation of p53 at Serine 15 is decreased in the presence of epirubicin after pre-treatment with vorinostat in a time- and concentration-dependent manner. Total p53 protein levels are also decreased, while mRNA levels remain unchanged after combined treatment. Treatment of breast cancer cells with valproic acid or entinostat also attenuates phosphorylation of p53 after epirubicin treatment, suggesting the involvement of Class I HDACs in the activation of p53. Phosphorylation of DNA repair kinase ATM is also decreased following DNA damage in the presence of clinically relevant concentrations of vorinostat, and corresponds with a decrease in activation of downstream factors including CHK1, CHK2, and BRCA1. These results demonstrate that inhibition of Class I HDACs in breast cancer cells delays DNA damaging response signaling through p53 modification after anthracycline administration; suggesting a possible mechanism for synergy between HDAC inhibitors and DNA damaging agents.

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 2614. doi:10.1158/1538-7445.AM2011-2614