The Regulatory T Cell–Associated Transcription Factor FoxP3 Is Expressed by Tumor Cells Lisa M. Ebert, 1 Bee Shin Tan, 1 Judy Browning, 1,2 Suzanne Svobodova, 1 Sarah E. Russell, 1 Naomi Kirkpatrick, 1 Craig Gedye, 1 Denis Moss, 3 Sweet Ping Ng, 1,2 Duncan MacGregor, 2 Ian D. Davis, 1 Jonathan Cebon, 1 and Weisan Chen 1 1 Ludwig Institute for Cancer Research (Melbourne Center for Clinical Sciences) and 2 Department of Pathology, Austin Health, Heidelberg, Victoria, Australia and 3 Epstein-Barr Virus Laboratory, Queensland Institute of Medical Research, Herston, Queensland, Australia Abstract FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25 + regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo , suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines. [Cancer Res 2008;68(8):3001–9] Introduction The immune system is subject to many levels of control, and distinct populations of T cells with ‘‘regulatory’’ (or suppressor) function make a major contribution to such regulation (1, 2). The best understood is the population of regulatory T cells (known as Treg), which are characterized by constitutive expression of CD25 and the transcription factor FoxP3 (3, 4). Despite an essential role in preventing autoimmunity, however, Treg may also have a negative effect on health by down-regulating beneficial immune responses, such as those mounted against tumors. Numerous animal studies have shown that removing or inhibiting Treg dramatically improves tumor clearance and survival (5, 6). Furthermore, several reports have documented the presence of Treg within human tumor tissue, the frequency of which may negatively correlate with survival (5–9). Thus, Treg may play a major role in preventing the development of effective antitumor immunity. FoxP3 is a member of the forkhead family of transcription factors and, at least in mice, seems to act as a ‘‘master switch’’ for the development and function of Treg (4, 10). Mice lacking functional expression of FoxP3 completely lack Treg and develop severe autoimmunity. Moreover, ectopic expression of FoxP3 in conventional murine T cells endows them with the full phenotype and function of Treg. In humans, there is also a strong association between FoxP3 expression and the Treg phenotype, although the relationship is more complex than in mice (10). For example, expression of FoxP3 can be transiently induced in human non-Treg cells by activation through the T-cell receptor (11–13). Never- theless, FoxP3 does seem to play a critical role in human Treg activity, as mutations in FOXP3 are associated with development of the multiorgan autoimmune disorder immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (14), in which Treg from affected patients have greatly reduced suppressive activity (15). Furthermore, ectopic expression of FoxP3 allows conventional human T cells to acquire many characteristics of Treg, although some studies have shown that their suppressive activity is inferior to that of bona fide Treg (16, 17). Until recently, FoxP3 expression has been thought to be restricted to the T-cell lineage. In the present study, however, we provide several lines of evidence that FoxP3 is also expressed by tumor cells. Given the central contribution of FoxP3 to Treg function, the expression of FoxP3 by tumor cells may represent a novel mechanism by which cancers suppress the immune system to escape destruction. Materials and Methods Cells, tissue, and media. Complete medium (RF-10) consisted of RPMI supplemented with 2 mmol/L Glutamax, 100 units/mL penicillin, 100 Ag/mL streptomycin, and 10 mmol/L HEPES (Invitrogen) and 10% FCS (Thermo Trace). Melanoma cell lines (LM-Mel series) and glioma cell lines (LM-G-2 and LM-G-4) were generated by culture of surgically excised tissue after mechanical and/or enzymatic disruption. Additional melanoma cell lines (SK-Mel-14 and SK-Mel-28), as well as renal cell carcinoma lines (SKRC-01, SKRC-09, and SKRC-017), were obtained from the Memorial Sloan-Kettering Cancer Center. The following additional cell lines were obtained from American Type Culture Collection: A172 and U-87MG (glioma); DU 145, PC-3, and LN-CaP (prostate cancer); A549, CaLu-6, and NCI-H460 (lung cancer); HCT116, caco-2, and SW480 (colorectal cancer); HT-1376, HT-1197, and HT-5637 (bladder cancer); MCF7, MDA-MB-231, and MDA-MD-468 (breast cancer). All tumor cell lines, as well as primary fibroblast cultures (derived from normal human dermis or foreskin), were maintained in RF-10 and passaged using trypsin/EDTA (Invitrogen) or 2 mmol/L EDTA in PBS. Normal epidermal melanocyte cultures were Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Cebon and W. Chen contributed equally to this work. Requests for reprints: Weisan Chen or Lisa Ebert, Ludwig Institute for Cancer Research, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australia. Phone: 61-3-9496-3700; Fax: 61-3-9457-6698; E-mail: weisan.chen@ludwig.edu.au or lisa.ebert@ludwig.edu.au. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-5664 www.aacrjournals.org 3001 Cancer Res 2008; 68: (8). April 15, 2008 Research Article Research. on November 21, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from