0041-1337/03/7603-597/0 TRANSPLANTATION Vol. 76, 597–602, No. 3, August 15, 2003 Copyright © 2003 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. TACROLIMUS ENHANCES TRANSFORMING GROWTH FACTOR- 1 EXPRESSION AND PROMOTES TUMOR PROGRESSION MARY MALUCCIO, 1 VIJAY SHARMA, 2 MILA LAGMAN, 2 SHEFALI VYAS, 2 HUA YANG, 2 BAOGUI LI, 2 AND MANIKKAM SUTHANTHIRAN 2,3 Background. Immunosuppressive therapy is a risk factor for the increased incidence and metastatic pro- gression of malignancies in organ graft recipients. Transforming growth factor (TGF)- 1 has been associ- ated with tumor invasion and metastasis, and we have implicated cyclosporine-associated TGF- 1 hyperex- pression in tumor progression in mice. Methods. BALB/c mice or severe combined immuno- deficient-beige mice were treated with 2 or 4 mg/kg of tacrolimus, and the effect of treatment on mouse renal cancer cell pulmonary metastasis was investigated. We also determined whether tacrolimus induces TGF- 1 expression. Spleens from tacrolimus-treated mice were analyzed for level of expression of TGF- 1 mRNA with the use of competitive-quantitative poly- merase chain reaction assay, and circulating levels of TGF- 1 protein were measured with the use of an en- zyme-linked immunosorbent assay. Results. Treatment with tacrolimus resulted in a dose- dependent increase in the number of pulmonary metasta- ses in the BALB/c mice (19716 in untreated mice, 28126 in mice treated with 2 mg/kg of tacrolimus, and 33925 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, Bonferroni’s P<0.001) and in the severe combined immunodeficient-beige mice (11718 in un- treated mice, 13719 in mice treated with 2 mg/kg of ta- crolimus, and 21629 in mice treated with 4 mg/kg of ta- crolimus; no treatment vs. 4 mg/kg tacrolimus, P<0.05). Treatment with 4 mg/kg but not 2 mg/kg of tacrolimus resulted in a significant increase in the levels of expression of TGF-  1 mRNA and circulating levels of TGF-  1 protein. Conclusions. Tacrolimus has a dose-dependent ef- fect on tumor progression and TGF- 1 expression, and tacrolimus-induced TGF- 1 overexpression may be a pathogenetic mechanism in tumor progression. Transforming growth factor (TGF)- 1 , a pleiotropic cyto- kine and secretory product of several cell types, has been implicated in many aspects of cancer biology including heightened invasiveness and metastatic progression (1, 2). The incidence and aggressiveness of certain malignancies are increased in organ graft recipients. Immunosuppressive drugs, by virtue of their ability to impair host immunosur- veillance mechanisms, are considered to be major risk factors for the heightened incidence and invasiveness of these ma- lignancies (3–6). In an earlier study, we identified a novel and host immu- nity-independent mechanism for the immunosuppressive drug cyclosporine-associated tumor progression (7). We found that cyclosporine conditioning of tumor cells conferred an invasive phenotype by a cell-autonomous mechanism and promoted neoplastic progression in T-cell, B-cell, and natural killer (NK)-cell– deficient severe combined immunodeficient (SCID)-beige mice. We also found that TGF- 1 blockade pre- vented cyclosporine-associated increase in the number of pul- monary metastases in the SCID-beige mice. Cyclosporine and tacrolimus are believed to exert their immunosuppressive effects through targeted binding and in- activation of calcineurin, a calcium- and calmodulin-depen- dent serine and threonine phosphatase (8, 9). Cyclosporine and tacrolimus have been shown to share not only immuno- suppressive mechanisms but also toxic side effects. Indeed, preclinical and clinical data exist indicating that tacrolimus, in a similar fashion to cyclosporine, is associated with renal fibrosis (10, 11). We reported that cyclosporine induces TGF- 1 overexpres- sion in multiple cell types (12, 13). We also observed that cyclosporine enhances in vivo expression of TGF- 1 in mice and humans (14, 15). Khanna et al. reported that in vitro conditioning of human T cells or human A-549 adenocarci- noma cells with tacrolimus results in the overexpression of TGF- 1 (16). Because TGF- 1 can enhance tumor invasive- ness and metastasis (1, 2), and because tacrolimus may share with cyclosporine the property of inducing TGF- 1 in vivo, we explored the in vivo effect of tacrolimus on tumor progression. In the current investigation, we used a well-established mouse renal cancer cell (RCC) pulmonary metastasis model (17) that shares several similarities with the clinical presen- tation of renal cancer to explore the effect of tacrolimus. Tacrolimus is a potent immunosuppressant and can impair host immunosurveillance barriers to tumor progression. To investigate the effect of tacrolimus on tumor progression independent of its effects on adaptive immunity, we also examined the effect of tacrolimus on tumor progression in T-, B-, and NK-cell– deficient SCID-beige mice (18). In view of the potential contribution of TGF- 1 overexpression to tumor progression, we investigated whether tacrolimus induces TGF- 1 overexpression in mice. MATERIALS AND METHODS Mice and Reagents Inbred male BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA) and were used at 6 to 8 weeks of age. Supported in part by a National Institutes of Health NIAID award (RO1 AI26932) and by an unrestricted award from Fujisawa Health- care, Inc. Dr. Mary Maluccio is a recipient of the Individual National Research Award (CA75732) from the National Institutes of Health. 1 Department of Surgery, Memorial Sloan Kettering Cancer Cen- ter, New York, New York. 2 Division of Nephrology, Department of Medicine, Department of Transplantation Medicine, Weill Medical College of Cornell Univer- sity, New York-Presbyterian Hospital, New York, New York. 3 Address correspondence to: M. Suthanthiran, M.D., 525 East 68th Street, Box 3, New York, NY 10021. E-mail: msuthan@med.cornell.edu. Received 27 February 2003. Revised 28 April 2003. Accepted 20 May 2003. 597 DOI: 10.1097/01.TP.0000081399.75231.3B