activates the phosphatase calcineurin. Activated calcineurin dephosphorylates NFAT, which then translocates to from the cytoplasm to the nucleus [12]. NFAT is a multigene family containing five members: NFAT (NFATc1-c5). Except for NFAT5, which is activated in response to osmotic stress [13], all NFAT family members are regulated by the calcium-activated protein phosphatase calcineurin and exist as transcriptionally inactive, cytosolic phosphoproteins [12]. There is increasing data supporting a critical role of NFAT in mediating angiogenic responses [1–3]. Importantly, NFAT activation was identified as a critical component of VEGF-induced angiogenesis and linked to the induction of cyclooxygenase-2 [14], which is also a critical player in angiogenesis. Our data suggested that NFAT may also mediate SFRP2 induced angiogenesis, as treatment of endothelial cells with SFRP2 resulted in an increase in nuclear NFATc3 [5]. In this study we further elucidate the role of both beta catenin and NFATc3 in SFRP2 mediated angiogenesis through RNA silencing, which confirms that NFATc3 is required for SFRP2 induced angiogenesis, while beta catenin is not. Thus, targeting NFAT with a calcineurin inhibitor may be a therapeutic strategy to inhibit both VEGF and SFRP2 induced angiogenesis. Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophlin FKBPB12, and the FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, resulting in inhibition of nuclear translocation of NFAT [1]. Tacrolimus is FDA approved for the prevention of organ transplant rejection and acts by inhibiting NFAT in lymphocytes [15]. Since FKBP12 has been reported to be expressed in benign and malignant vascular endothelium [16], we hypothesize that FKBP12 is expressed in breast tumor endothelium, allowing tacrolimus to inhibit breast tumor angiogenesis and tumor growth. Materials and Methods Treatment of MMTV-neu transgenic mice with tacrolimus in vivo This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of North Carolina at Chapel Hill, IACUC ID# 09-134.0. We had previously shown that treatment with tacrolimus 3 mg/kg/day intraperitoneal (i.p.) was effective at suppressing the growth of SVR angiosarcoma tumor in nude mice as compared with control by 46% without signs of toxicity [5]. Using an FDA Oncology Calculator, we calculated this dose to be equivalent to the human dose of 0.24 mg/kg/day [5], which is the dose that is used to prevent human liver transplant rejection. To evaluate whether tacrolimus would inhibit the growth rate of breast tumors in vivo, we studied the efficacy of tacrolimus on the growth of MMTV-neu tumors in transgenic mice. Female FVB/ N-Tg(MMTVneu) 202Mul/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, Stock Number 002376). The experimental female mice were bred in order to speed the onset of tumorigenesis and were housed and inspected for tumor development twice weekly. All animal work was conducted in UNC DLAM animal facilities under an approved IACUC protocol. Once tumors were detected by palpation or visual inspection, tumor volume was documented using calipers and tumor volume was calculated using the following formula: (shortest diameter)26(longest diameter)60.52. When tumors reached 100– 3 300 mm , mice were treated with either tacrolimus 3 mg/kg/day i.p. in 20% Intralipid (Baxter, Deerfield, IL) or control (which consisted of no treatment n = 9 or 20% Intralipid i.p. n = 6). All Tacrolimus Inhibits Breast Cancer Growth In Vivo tumors that were present from day 1 were measured. Mice used in this study developed either one or two mammary tumors. Tumor growth (assessed as percent change in tumor volume over initial tumor volume) was calculated using the formula: [(final volu- me2initial volume)/initial volume6100]. Antibodies The following antibodies were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA: FKBP12 (sc-28814), NFTAc3 (sc- 8405), SFRP2 (sc-13940), and b-catenin (sc-59893). The loading control, TATA binding protein TBP antibody (ab818), was purchased from Abcam, Inc. (Cambridge, MA). CD31 primary antibody was purchased from NeoMarkers (Fremont, CA). Secondary antibodies were purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ): ECL anti-mouse IgG, HRP-linked whole antibody (NA931) and ECL anti-rabbit IgG, HRP-linked whole antibody (NA934). Immunohistochemistry CD31 staining in mouse breast tumors. To show whether the reduced growth rate of tacrolimus treated tumors correlates with a decrease in tumor angiogenesis, we sectioned 4 control tumors from mice treated with 20% intralipid and 4 tumors from mice treated with tacrolimus. Breast tumors were sectioned at 5 mM onto Superfrost plus slides. Slides were dewaxed by immersing in xylene three times for 5 minutes each. Slides were hydrated in 100% ETOH, 95% ETOH, 70% ETOH, and 50% ETOH for 3 minutes each. Slides were quenched in 3% H2O2 (DakoCytomation, LSAB2 HRP Kit, Carpinteria, CA) for 10 minutes, and then PBS for 3 minutes. Citra buffer (BioGenex, San Ramon, CA) was warmed in a 60uC oven and slides were immersed in citra buffer at 100uC in a rice steamer for 30 minutes. Slides were rinsed in PBS for 3 minutes and then marked with a PAP pen. Goat serum (Pierce Biotechnology, Rockford, IL) block (3 mg/ml) was applied for 10 minutes at room temperature. Slides were rinsed twice in PBS for 3 minutes then CD31 primary antibody (250 ml at 1:100 dilution) was applied and slides were placed in a covered box in a 4uC cold room overnight. Slides were then rinsed in PBS for 3 minutes, and 1–2 drops of biotinylated secondary antibody (Ultravision Detection System, LabVision Corp, Fremont, CA) was added to each slide for 20 minutes. Slides were rinsed twice in PBS for 3 minutes and 1–2 drops of streptavidin-HRP (Ultravision Detection System, LabVision Corp, Fremont, CA) was applied for 20 minutes. One-two drops of DAB complex (Ultravision Detection System, LabVision Corp, Fremont, CA) was applied to each slide for approximately 10 minutes. Slides were rinsed twice in distilled water for 3 minutes each and counterstained with trypan blue (Sigma, St Louis, MO) for 5 minutes. Slides were rinsed in PBS, dehydrated through graded alcohol and xylene, and Cytoseal XYL (Richard-Allan, Kalamazoo, MI) and cover slides were applied. A negative control without primary antibody was performed for all experiments. Slides were evaluated for the presence of CD31 staining in tumor endothelium.Tumor vascularity was quantified as described previously to identify the number of microvessels/unit area (6200) [17–20]. The mean of three fields judged to have the greatest numbers of microvessels was used for comparison between control and tacrolimus treated tumors. FKBP12 staining in human breast tumors. Research involving archival human samples was approved by the IRB at the University of North Carolina at Chapel Hill on an IRB approved protocol # 05-2442. A waiver for obtaining consent was obtained because the research involved only existing human PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20412