483 A Novel Metabolic Anti-cancer Therapy – Targeting Liver Tumors Through Mitochondrial Damage

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european journal of cancer 48, suppl. 5 (2012) S25–S288

For more in-depth characterization of the heterogeneity of the CSC population we have by quantitative proteomics and transcriptomic analysis investigated single cell clones that are able to initiate tumor, establish mammospheres, and have an epithelial morphology and a CD24low profile in contrast to another clone with a mesenchymal-like morphology, a CD24− profile and the inability to establish tumors and mammospheres. The selected clones have led to discovery of a number of proteins/genes that could lead to the investigation of new biomarkers characterizing CSCs in different phenotypic stages. Conclusions: We propose that the CD44+ /CD24−/low profile is not a sufficient marker for breast CSCs. Our work have led to knowledge of the characteristics of single cell clones that were either CSC-like, non-CSC-like or intermediate, thus tumor initiating capabilities in vivo are the most profound CSC characteristic. 482 Acid-extruding Proteins as Potential Novel Targets in Human Breast Cancer S.F. Pedersen1 , G. Lauritzen1 , E. Boedtkjer2 , A. Gorbatenko1 , J. Moreira3 , C.W. Olesen1 , C.M. Stock4 , C. Aalkjaer2 . 1 University of Copenhagen, Department of Biology, Copenhagen, Denmark, 2 Aarhus University, Department of Biomedicine, Aarhus, Denmark, 3 Danish Cancer Society, ¨ Genome Integrity Unit, Copenhagen, Denmark, 4 University of Munster, Physiologisches Institut-II, Munster, ¨ Germany Introduction: A fundamental property of solid tumors is a markedly altered pHprofile compared to normal tissues, with acidic extracellular, and often alkaline intracellular pH (pHi ). This at least in part reflects the increased glycolytic metabolism of tumor cells, necessitating increased acid extrusion to maintain survival. Acid extruding transporters are therefore potentially interesting targets in cancer. The aim of the studies presented here was to explore the roles and regulation of the net acid extruding transporters, the Na+ /H+ exchanger NHE1 and the Na+ , HCO−3 cotransporter NBCn1, in human breast cancer. Materials and Methods: A combination of cell biological and molecular biology approaches were employed, including immunoblotting, immunofluorescence, siRNA mediated knockdown, pH-sensitive fluorescent probes, adhesion and migration assays, as well as immunohistochemistry on patient tissue samples. Results and Discussion: Expression of a constitutively active, truncated form of the ErbB2/HER2 receptor DNErbB2) greatly increased pH regulatory capacity in MCF-7 breast cancer cells, in a manner mediated by NHE1 and NBCn1 [1]. After DNErbB2 expression or stimulation of wild-type ErbB receptors, NBCn1 was strongly upregulated at the mRNA and protein levels, and NHE1 was phosphorylated at Ser703 , a known target for the 90 kDa Ribosomal S6-Kinase (p90RSK). In MCF-7 cells, NHE1 was strongly expressed in invadopodial rosettes, colocalizing with the invadopodial markers cortactin, F-actin and phospho-Tyr416-Src, as well as with its known binding partners, ezrin and radixin [2]. NHE1 inhibition or siRNA-mediated NHE1 knockdown potently sensitized DNErbB2-MCF-7 cells to cisplatin-induced apoptosis, whereas it enhanced their adhesion and 2D migration on collagen-I [1]. Finally, using matched sets of patient tissue we show that NHE1 and NBCn1 are upregulated in primary breast carcinomas and lymph node metastases compared to normal breast tissue, and we demonstrate that Na+ , HCO−3 cotransport is a major determinant of pHi regulation in freshly dissected human breast tumors [3]. Conclusion: Expression and regulation of the two major pH regulatory ion transporters NHE1 and NBCn1 are altered in human breast cancer, resulting in altered pH regulation. NBCn1 expression is strongly sensitive to ErbB receptor activity, and NHE1 regulates chemotherapy sensitivity and cell motility. This suggests that NHE1 and NBCn1 are potential targets in breast cancer diagnosis and/or treatment. Reference(s) [1] Lauritzen et al. 2010 Exp Cell Res 316, 2538–2553 [2] Lauritzen et al. 2012 Cancer Letters 317(2):172−83 [3] Boedtkjer et al. 2012 Int J Cancer, submitted. Funding: Danish Cancer Society, Novo Foundation, Lundbeck Foundation. 483 A Novel Metabolic Anti-cancer Therapy − Targeting Liver Tumors Through Mitochondrial Damage V. Tesori1 , A.C. Piscaglia1 , M. Barba1 , M.A. Puglisi1 , G. Pani2 , G. Gasbarrini3 , A. Gasbarrini1 . 1 Catholic University of the Sacred Heart, Internal Medicine and Gastroenterology, Rome, Italy, 2 Catholic University of the Sacred Heart, General Pathology, Rome, Italy, 3 Catholic University of the Sacred Heart, Research in Medicine Foundation, Rome, Italy Introduction: One of the basis of modern oncology resides on the metabolic alteration of cancer cells, that explicates through an increase of glycolytic pathway, the so called ‘Warburg Effect’. Glycolysis can be seen as a cancer antioxidant response to allow tumor dissemination protecting cells from ROSinduced senescence and apoptosis. Although the multikinase inhibitor Sorafenib specifically targets tumor cells harboring the V590E mutation of B-RAF, additional mechanisms likely account

Sunday 8 − Tuesday 10 July 2012

for the elevated anticancer activity of the drug in tumors lacking this mutation, and for the emergence of chemoresistance. Aim: In the present study a novel metabolic effect of the multikinase inhibitor Sorafenib (SFB) was investigated, alone and in combination with the glycolytic inhibitor 2-deoxyglucose (2dg), in a liver tumor model, the LCSC-2 (Liver Cancer Stem Cells) cell line. In particular we investigated the mitochondrial action of SFB. Material and Methods: Drug toxicity was evaluated through Propidium Iodide (PI) assay. Gene expression was investigated by microarray analysis. Protein expression was evaluated by western blotting. Intracellular ATP level was assessed by chemiluminescence. Mitochondrial activity was assessed by Respirometric Analysis. Mitochondrial potential was measured with the JC-1 probe. Results and Discussion: We found that in rat hepatocolangiocarcinoma (LCSC-2) cells exposure to Sorafenib was not paralleled by significant inhibition of ERK phosphorylation, but, instead, elicited a raise of intracellular reactive oxygen species and the Ser172 phosphorylation of the AMP-activated protein kinase (AMPK), two events consistent with mitochondrial dysfunction. Accordingly, SFB led to a substantial reduction of oxygen consumption, cellular ATP level and mitochondrial transmembrane potential, and isolated mitochondria from rat liver were depolarized by the drug in vitro, indicating a direct effect of SFB on the organelle. Interestingly, in keeping with its inhibitory action on mitochondrial respiration, Sorafenib killed much more efficiently LCSC-2 cells, as well as mouse B16F10 melanoma and 293T human kidney carcinoma cells when associated with the glycolysis inhibitor 2-deoxyglucose. Conclusions: Taken together our preliminary results identify in mitochondrial damage and generation of ROS a novel modality of cytotoxicity by Sorafenib; moreover, the synergistic action of SFB plus 2DG outline a novel combined therapy to eradicate liver tumors. 484 Protection Against ER Stress in the Tumor Microenvironment by Loss of P19ARF S. Ryeom1 , D. Crawford1 , K. Stewart1 . 1 University of Pennsylvania School of Medicine, Cancer Biology, Philadelphia PA, USA Cellular stress occurs during tumorigenesis due to a number of factors including an increase in reactive oxygen species, restricted nutrients and low O2 levels or hypoxia. More recently, endoplasmic reticulum (ER) stress has emerged as a major player in tumor pathology as a consequence of hypoxia, among other triggers. Although cells in the tumor microenvironment are considered to be more epigentically stable than the tumor cells, they are exposed to the same cellular stresses as tumor cells. While the impact of these stresses on tumor cells have been well-studied, the effects on other cells in the tumor microenvironment remains to be elucidated. Here, we examine the effect of hypoxia-induced ER stress in the tumor vasculature and demonstrate activation of the Unfolded Protein Response (UPR) in the endothelium. The UPR is generally thought to be cytoprotective allowing cells to recover from stress through activation of the PERK pathway which globally halts translation through phosphorylation of the eukaryotic translation initiation factor 2a (eIF2a). However, after prolonged or excessive levels of stress, the UPR will trigger apoptosis. Here we show that the tumor suppressor p19ARF is activated in endothelial cells subjected to hypoxia or pharmacologicallyinduced ER stress leading to endothelial cell cycle arrest via p53-independent mechanisms. p19ARF has been well characterized to be induced by oncogenic activation in tumor cells and stabilizes p53 via sequestration of Mdm2 in the nucleolus thus activating a tumor suppressive response. Our data shows that transactivation of p19ARF in endothelial cells occurs by E2F1 which is induced during ER stress and that ER stress-mediated activation of p19ARF leads to inhibition of ribosomal RNA processing in the nucleolus, leading to cell cycle arrest and/or apoptosis. Further, loss of p19ARF in endothelial cells confers resistance to ER stress-induced cell cycle arrest and apoptosis resulting in endothelial cell proliferation regardless of p53 status. Taken together, our data suggests that there is a selective pressure for endothelial cells in the tumor microenvironment that have lost p19ARF expression. p19ARF−/− endothelial cells are thus able to survive and proliferate in the presence of ER stress, leading to increased angiogenesis and ultimately promoting tumor growth. Therefore, ER stress represents a novel inducer of p19ARF and could provide targets for anti-angiogenic therapy independent of VEGF inhibition. 485 Distinct Recycling of Active and Inactive b1 Integrins A. Arjonen1 , J. Alanko1 , S. Veltel1 , J. Ivaska1 . 1 University of Turku, Centre for Biotechnology, Turku, Finland Background: Cancer cell migration is critically co-ordinated by trafficking of plasma membrane receptors. Integrins are a large family of heterodimeric cell surface adhesion receptors consisting of 24 non-covalently associated a and b subunits. Integrins bind to extracellular matrix ligands such as collagens and fibronectin. Integrin-containing adhesions allow cells to exert adhesion forces between different parts of the cell body, which is a prerequisite for