Ceramide Synthase 1–Regulated Sensitivity to Cisplatin Is Associated
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(Dihydro)ceramide Synthase 1–Regulated Sensitivity to Cisplatin Is Associated with the Activation of p38Mitogen-Activated Protein Kinase and Is Abrogated by Sphingosine Kinase 1 Junxia Min,1 Adi Mesika,3 Mayandi Sivaguru,1,2 Paul P. Van Veldhoven,4 Hannah Alexander,1 Anthony H. Futerman,3 and Stephen Alexander1 1Division of Biological Sciences and 2Molecular Cytology Core, University of Missouri, Columbia, Missouri; 3Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel; and 4Katholieke Universiteit Leuven, Departement Moleculaire Celbiologie, Afdeling Farmakologie, Leuven, Belgium Abstract LASS4/CerS4 or LASS5/CerS5, from the endoplasmic Resistance to chemotherapeutic drugs often limits their reticulum (ER) to the Golgi apparatus. Supporting the clinical efficacy. Previous studies have implicated the hypothesis that this translocation is mechanistically bioactive sphingolipid sphingosine-1-phosphate (S-1-P) involved in the response to cisplatin, we showed that in regulating sensitivity to cisplatin [cis-diamminedi- expression of SphK1, but not SphK2, abrogates both the chloroplatinum(II)] and showed that modulating the increased cisplatin sensitivity in cells stably expressing S-1-P lyase can alter cisplatin sensitivity. Here, we show LASS1/CerS and the translocation of the LASS1/CerS1. that the members of the sphingosine kinase (SphK1 and The data suggest that the enzymes of the sphingolipid SphK2) and dihydroceramide synthase (LASS1/CerS1, metabolic pathway can be manipulated to improve LASS4/CerS4, and LASS5/CerS5) enzyme families each sensitivity to the widely used drug cisplatin. have a unique role in regulating sensitivity to cisplatin (Mol Cancer Res 2007;5(8):801–12) and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and Introduction vincristine, whereas expression of SphK2 increases Chemotherapy is frequently used in the treatment of cancer, sensitivity. Expression of LASS1/CerS1 increases the but drug resistance greatly limits the efficacy of treatment. sensitivity to all the drugs tested, whereas LASS5/CerS5 Some cancers are initially resistant to chemotherapy, whereas only increases sensitivity to doxorubicin and vincristine. others become resistant during the course of treatment. This is LASS4/CerS4 expression has no effect on the sensitivity true for the drug cisplatin [cis-diamminedichloroplatinum(II)], to any drug tested. Reflecting this, we show that the which is widely used to treat a variety of solid tumors including activation of the p38mitogen-activated protein (MAP) non-Hodgkin’s lymphoma, small cell and non–small cell lung kinase is increased only by LASS1/CerS1, and not by cancers, testicular, ovarian, head and neck, esophageal, and LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to bladder cancers (1). Many different mechanisms of resistance to cause a specific translocation of LASS1/CerS1, but not cisplatin have been investigated; yet it is clear that the resistance to cisplatin is complex and multifaceted, and we are far from having a full understanding of all the relevant Received 2/27/07; revised 5/31/07; accepted 6/12/07. Grant support: Work in the Alexander lab is supported by NIH grant GM53929. signaling pathways involved (2). A better understanding of the Work in the Futerman lab is supported by the Israel Science Foundation, grant mechanisms controlling the sensitivity to cisplatin could be 1047/03, and by the Minerva Foundation, Munich, Germany. A.H. Futerman is used to make the drug more effective. the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. S. Alexander and A.H. Futerman are the corecipients of United States – In an unbiased genetic examination of genes that are Israel National Science Foundation grant. P.P. Van Veldhoven was supported by involved with the cellular response and sensitivity to cisplatin grants from the Flemish Fonds voor Werenschappelijk Onderzoek (G.405.02) and from the Belgian Ministry of Federaal Wetenschapsbeleid Interuniversitarie in the model eukaryote Dictyostelium discoideum, we showed Attractiepolen (IAP-P5/05). that disruption of the gene encoding sphingosine-1-phosphate The costs of publication of this article were defrayed in part by the payment of (S-1-P) lyase resulted in decreased sensitivity to the drug (3). page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This defined the S-1-P lyase as a potential molecular target for Note: Supplementary data for this article is available at Molecular Cancer controlling sensitivity to cisplatin and suggested a general role Research Online (http://mcr.aacrjournals.org/). for sphingolipids in the cellular response to the drug. S-1-P is Current address for J. Min: Brigham and Woman’s Hospital, Department of Medicine/Division of Genetics, Harvard Medical School New Research Building, synthesized from sphingosine and ATP by two distinct 77 Avenue Louis Pasteur, Boston, MA 02115. sphingosine kinases, and S-1-P lyase catalyzes the conversion Current address for M. Sivaguru: Institute for Genomic Biology, 1206 W. Gregory Dr., University of Illinois, Urbana, IL 61801. of S-1-P to hexadecenal and phosphoethanolamine at the end of Requests for reprints: Stephen Alexander, Division of Biological Sciences, 303 the pathway of sphingomyelin metabolism (Fig. 1). S-1-P is a Tucker Hall, University of Missouri, Columbia, MO 65203. Phone: 573-882- bioactive sphingolipid that is involved in many cellular 6670; Fax: 573-882-0123. E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. functions including controlling cell proliferation, cell differen- doi:10.1158/1541-7786.MCR-07-0100 tiation, and cell movement (4). The prevailing thought was that Mol Cancer Res 2007;5(8). August 2007 801 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. 802 Min et al. FIGURE 1. Pathway of sphingolipidmetabolism. The enzymes of special importance to this studyare the sphingosine kinases ( SphK1-2) andthe dihydroceramide synthases (CerS1-6/LASS1-6). Previous studies had shown that high ceramide promotes cell death, whereas high S-1-P promotes cell proliferation. The current study reveals a more detailed picture. DH, dihydro, PEA, phosphoethanolamine. S-1-P functions in a rheostat-like mechanism with another to ask whether modulation of any, or all, of these enzymes bioactive sphingolipid, ceramide, where the relative levels of results in an altered cellular response to cisplatin, and to begin the two sphingolipids control whether cells proliferate (high to establish the roles of these enzymes relative to one another in S-1-P) or die (high ceramide; refs. 4, 5). This, coupled with the controlling this response. Synthesis of S-1-P in human cells is observed decreased cisplatin sensitivity in the S-1-P lyase null mediated by two sphingosine kinase enzymes, which are mutant, led us to suggest that modulating the levels of the reported to have opposing roles in the cell (13). Sphingosine enzymes for the synthesis and degradation of S-1-P would have kinase 1 (SphK1) is a prosurvival enzyme, which promotes cell predictable effects on the sensitivity to cisplatin (6). Indeed, proliferation, whereas sphingosine kinase 2 (SphK2) is believed extensive genetic, biochemical, and pharmacologic studies in to be proapoptotic. The SphK2 protein contains a domain with D. discoideum further established a clear role for the S-1-P homology to the BH3-only proteins, common to the proapop- lyase as well as the sphingosine kinases in controlling cisplatin totic members of the Bcl-2 family of proteins (14). Both SphK1 sensitivity, where cells overexpressing sphingosine kinase or and SphK2 use the natural substrate D-erythro-sphingosine with null for S-1-P lyase have decreased sensitivity to cisplatin, similar Km values (although SphK2 can use a broader range of whereas cells null for the sphingosine kinase or overexpressing analogs; refs. 15-17). This suggests the possibility that it is the S-1-P lyase are more sensitive. The change in sensitivity cellular location and/or specific protein-protein interactions of was seen with cisplatin and carboplatin, but not with other these enzymes that determines their effect on the cell, rather drugs (6-8). than the absolute level of S-1-P in the cell (13). These studies were extended to human cells. We showed The de novo synthesis of the upstream bioactive molecule increased drug sensitivity in either HEK293 cells (human ceramide in human cells is executed by a family of six embryonic kidney) or A549 cells (human lung cancer) over- transmembrane dihydroceramide synthase enzymes that are expressing the human S-1-P lyase gene, and again, the response involved in the synthesis of dihydroceramide from sphinganine was highly biased toward the platinum-based drugs cisplatin and fatty acyl CoA. These enzymes were originally termed and carboplatin. Moreover, we showed that inhibition of the LASS (longevity assurance) genes to denote that the first gene sphingosine kinase with the competitive inhibitor dimethyl- discovered in yeast (LAG1) extended life span (18), but they sphingosine has a synergistic effect on increasing cisplatin have been renamed CerS to more closely reflect their sensitivity (9). Mechanistically, we showed that the increase in biochemical nature (19). Accumulating evidence suggests that cisplatin sensitivity resulting