Tumor-Associated Antigen Preferentially Expressed Antigen of Melanoma (PRAME) Induces Caspase-Independent Cell Death In vitro and Reduces Tumorigenicity In vivo Nicolas Tajeddine, 1 Jean-Luc Gala, 2 Magali Louis, 2 Monique Van Schoor, 1 Bertrand Tombal, 1 and Philippe Gailly 1 1 Laboratory of Cell Physiology and 2 Laboratory of Applied Molecular Technology, Center for Human Genetics, Universite´ catholique de Louvain, Brussels, Belgium Abstract Preferentially expressed antigen of melanoma (PRAME) is expressed in a wide variety of tumors, but in contrast with most other tumor associated antigens, it is also expressed in leukemias. The physiologic role of PRAME remains elusive. Interestingly, PRAME expression is correlated with a favorable prognosis in childhood acute leukemias. Moreover, a high expression of PRAME seems to be predominantly found in acute leukemias carrying a favorable prognosis. On these clinical observations, we assumed that PRAME could be involved in the regulation of cell death or cell cycle. In this study, we show that transient overexpression of PRAME induces a caspase-independent cell death in cultured cell lines (CHO-K1 and HeLa). Cells stably transfected with PRAME also exhibit a decreased proliferation rate due, at least partially, to an elevated basal rate of cell death. Immunocy- tochemistry of a FLAG-tagged PRAME, in vivo imaging of an enhanced green fluorescent protein–tagged PRAME, and Western blotting after cell fractionation reveal a nuclear localization of the protein. Using a microarray-based approach, we show that KG-1 leukemic cells stably transfected with PRAME present a significant decrease of expression of the heat-shock protein Hsp27, the cyclin-dependent kinase inhibitor p21, and the calcium-binding protein S100A4. The expression of these three proteins is known to inhibit apoptosis and has been associated with an unfavorable prognosis in a series of cancers. Finally, repression of PRAME expression by a short interfering RNA strategy increases tumorigenicity of K562 leukemic cells in nude mice. We suggest that all these observations might explain the favorable prognosis of the leukemias expressing high levels of PRAME. (Cancer Res 2005; 65(16): 7348-55) Introduction During the last couple of years, several classes of human tumor-associated antigens (TAA) recognized by autologous CTLs have been identified (1). An antitumor CTL response can be induced by antigens encoded by genes overexpressed in tumors. Preferentially expressed antigen of melanoma (PRAME) was identified as a gene encoding an HLA-A24–restricted antigenic peptide presented to an autologous tumor-specific CTL clone derived from a melanoma cell line called LB-33.MEL (2). PRAME encodes a putative protein of 509 amino acids, the function of which is still unknown. Whereas most normal tissues do not express PRAME, a very low expression was observed in testis, placenta, endometrium, ovary, and adrenals (1). PRAME expres- sion was found in several tissues and cell lines derived from melanoma, lung, kidney, and head and neck carcinomas. Unlike the other TAAs, PRAME was also expressed in adult as well as pediatric acute leukemias: in 40% to 60% of acute lymphoid leukemia (ALL) and in more than 60% of acute myeloid leukemia (AML) at diagnosis (3, 4). Interestingly, a high expression of PRAME seems to be predominantly found in acute leukemias carrying a favorable prognosis: AML with the (8;21) translocation, AML-M3 with the (15;17) translocation, and childhood B-ALL with or without the (12;21) translocation (5). In addition, overexpression of PRAME seems to be associated with signifi- cantly higher rates of overall and disease-free survival and lower relapse rate, compared with patients with no or low PRAME expression (4, 6). At least in one case, it has also been shown that leukemic cells are able to process the PRAME protein, to present its peptide on their cell surface by HLA-A24, and to be lysed by cytotoxic cells (7). Whereas those lines of evidence seemed to link prognosis and immune response in PRAME-positive malig- nancies, the unusually high response rate of t(8;21) and t(15;17) AML to chemotherapy suggested that nonimmune mechanisms may also determine the response to xenobiotics. The current study aims at evaluating the contribution of PRAME in the regulation of cell proliferation, programmed cell death, and tumorigenicity. Materials and Methods Cell Lines A panel of cell lines was chosen according to their basal level of endogenous PRAME expression: the Chinese hamster ovary (CHO) trans- formed fibroblasts CHO-K1 cell line and the acute myeloblastic leukemia cell line KG-1 express no or very low amounts of PRAME 3 whereas the HeLa cervical carcinoma and K562 chronic myelogenous leukemia cell lines express significant amounts of the protein. All cell lines were purchased from American Type Culture Collection (Manassas, VA). KG-1 and K562 cell lines were grown at 37jC in a 5% CO 2 humidified atmosphere in RPMI 1640 (Life Technologies, Inc., Carlsbad, CA). CHO-K1 and HeLa cell lines were grown in F12-Ham and DMEM media, respectively (Life Technologies). All media were supplemented with 100 mL/L FCS (Life Technologies) and 10 mL/L glutamine (Life Technologies). Note: N. Tajeddine and J-L. Gala contributed equally to this work. Requests for reprints: Philippe Gailly, Laboratory of Cell Physiology, Universite´ catholique de Louvain, Avenue Hippocrate, 55/40, 1200 Brussels, Belgium. Phone: 32-2-764-55-42; Fax: 32-2-764-55-80; E-mail: gailly@fycl.ucl.ac.be. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-04-4011 3 Unpublished data. Cancer Res 2005; 65: (16). August 15, 2005 7348 www.aacrjournals.org Research Article Research. on January 2, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from