Functional Interaction between Acyl-CoA Synthetase 4, Lipooxygenases and Cyclooxygenase-2 in the Aggressive Phenotype of Breast Cancer Cells Paula M. Maloberti 1 , Alejandra B. Duarte 1 , Ulises D. Orlando 1 , Marı ´a E. Pasqualini 2 ,A ´ ngela R. Solano 1 , Carlos Lo ´ pez-Otı´n 3 , Ernesto J. Podesta ´ 1 * 1 Instituto de Investigaciones Moleculares de Enfermedades Hormonales Neurodegenerativas y Oncolo ´ gicas (IIMHNO), Department of Human Biochemistry, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina, 2 Instituto de Biologı ´a Celular, School of Medicine, Co ´ rdoba National University, Co ´ rdoba, Argentina, 3 Instituto Universitario de Oncologı ´a, Department of Biochemistry and Molecular Biology, Oviedo University, Oviedo, Espan ˜a Abstract The acyl-CoA synthetase 4 (ACSL4) is increased in breast cancer, colon and hepatocellular carcinoma. ACSL4 mainly esterifies arachidonic acid (AA) into arachidonoyl-CoA, reducing free AA intracellular levels, which is in contradiction with the need for AA metabolites in tumorigenesis. Therefore, the causal role of ACSL4 is still not established. This study was undertaken to determine the role of ACSL4 in AA metabolic pathway in breast cancer cells. The first novel finding is that ACSL4 regulates the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin in MDA-MB-231 cells. We also found that ACSL4 is significantly up-regulated in the highly aggressive MDA-MB-231 breast cancer cells. In terms of its overexpression and inhibition, ACSL4 plays a causal role in the control of the aggressive phenotype. These results were confirmed by the increase in the aggressive behaviour of MCF-7 cells stably transfected with a Tet-off ACSL4 vector. Concomitantly, another significant finding was that intramitochondrial AA levels are significantly higher in the aggressive cells. Thus, the esterification of AA by ACSL4 compartmentalizes the release of AA in mitochondria, a mechanism that serves to drive the specific lipooxygenase metabolization of the fatty acid. To our knowledge, this is the first report that ACSL4 expression controls both lipooxygenase and cyclooxygenase metabolism of AA. Thus, this functional interaction represents an integrated system that regulates the proliferating and metastatic potential of cancer cells. Therefore, the development of combinatory therapies that profit from the ACSL4, lipooxygenase and COX-2 synergistic action may allow for lower medication doses and avoidance of side effects. Citation: Maloberti PM, Duarte AB, Orlando UD, Pasqualini ME, Solano A ´ R, et al. (2010) Functional Interaction between Acyl-CoA Synthetase 4, Lipooxygenases and Cyclooxygenase-2 in the Aggressive Phenotype of Breast Cancer Cells. PLoS ONE 5(11): e15540. doi:10.1371/journal.pone.0015540 Editor: Janine Santos, University of Medicine and Dentistry of New Jersey, United States of America Received July 26, 2010; Accepted October 22, 2010; Published November 11, 2010 Copyright: ß 2010 Maloberti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by CONICET (112-200801-01976)http://www.conicet.gov.ar/, Podesta ´ ; UBA (M059, M810)Podesta ´ , Maloberti, http://www.uba.ar/homepage.php; FONCyT (2006- 00819, 25368, 32527)Podesta, Podesta, Maloberti; http://www.agencia.mincyt.gov.ar/, Bunge & Born Foundation, Podesta, Solano, http://www.fundacionbyb.org/ ing_investigacion_cientifica.asp; and Ministerio de Ciencia e Innovacio ´ n-Spain, http://www.micinn.es/portal/site/MICINN/, Fundacio ´ n "M. Botı ´n",http://www. fundacionmbotin.org/, and European Union (FP7 MicroEnviMet)http://ec.europa.eu/research/health/medical-research/cancer/fp7-projects/microenvimet_en.html, Lopez Otı ´n. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ernestopodesta@yahoo.com.ar Introduction The acyl-CoA synthetase, ACSL4 or FACL4, belongs to a five- member family of enzymes that esterify mainly arachidonic acid (AA) into acyl-CoA [1,2]. A striking feature of ACSL4 is its abundance in steroidogenic tissues [1]. In contrast, ACSL4 is poorly expressed in other adult tissues, including breast, liver and the gastrointestinal tract in general [3,4,5]. Abnormal expression of ACSL4 in non-steroidogenic tissues has been involved in tumorigenesis [3,4,6]. In fact, ACSL4 overexpression has been reported in colon adenocarcinoma, hepatocellular carcinoma and breast cancer [3,4,5]. In human breast cancer, ACSL4 is differentially expressed as a function of estrogen receptor alpha status [5]. The release of AA has been indicated as an important signal leading to cellular proliferation. AA is, in turn, converted to different biologically active eicosanoid metabolites by three main enzymatic activities: lipooxygenase (LOX), cyclooxygenase (COX) and epooxygenase-cytochrome P450. LOX and COX are known to play a critical role in cancer progression i.e. growth and metastasis [7,8,9,10]. Differences in abundance and activity of AA-converting enzymes may result in variations in the cellular content of eicosanoids. Therefore, and in view of the potential effects exerted by AA and derived eicosanoids, the enzymatic release of AA, its intracellular distribution and concentration are all under rigorous control within cells. Classically, activation of cytosolic phospholi- pase A2 has been considered as the rate-limiting step in the generation of AA. However, an alternative pathway that releases AA in specific compartments of the cell, e.g. mitochondria, has been described in steroidogenic tissues [11,12,13,14]. This pathway, in which the rate-limiting enzyme is ACSL4, provides arachidonoyl-CoA to a mitochondrial acyl-CoA thioesterase (ACOT2) that releases AA in mitochondria and directs this fatty acid to the LOX enzyme for its subsequent conversion to lipooxygenase metabolites [15,16]. In this pathway, the Translo- PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15540