Tacrolimus Inhibits Breast Cancer Growth In Vivo (7 nM) and siRNA beta catenin transfected cells stimulated with SFRP2 (7 nM) [Fig. 3B]. This demonstrates that beta catenin is not required for SFRP2 induced tube formation. of NFATc3 with tacrolimus treatment (Figure 5B). This shows that tacrolimus inhibits SFRP2 induced NFATc3 translocation in endothelial cells without reducing total NFATc3 protein. NFATc3 is required for SFRP2 induced endothelial tube branching We have reported that endothelial cells treated with SFRP2 have an increase in nuclear NFATc3, and inhibition of NFAT in endothelial cells with the calcineurin inhibitor tacrolimus inhibits SFRP2 stimulated tube formation [5]. To definitively show whether NFATc3 is required for SFRP2 stimulated angiogenesis, we established a stable 2H11 endothelial cell line with shRNA to NFATc3, and Western blot analysis demonstrated that NFATc3 protein is decreased in shRNA-NFATc3 transfected cells by 69% compared to sham-transfected controls (Fig. 4A, Supplemental Fig. S2B). Cells were then seeded for a 6 hour tube formation assay. Sham transfected cells responded to SFRP2 stimulation with a statistically significant increase in the number of branch points (p,0.01) (Fig. 4B, Figure S3A). However, 2H11 cells transfected with shRNA to NFATc3 showed no increase in tube formation in response to SFRP2 stimulation (Fig. 4B, Figure S3B). This shows that NFATc3 is required for SFRP2 mediated endothelial tube formation. Tacrolimus inhibits SFRP2 activation of NFATc3 in endothelial cells To evaluate the role of tacrolimus on the non-canonical Wnt/ Ca++ pathway in SFRP2 induced angiogenesis, we compared nuclear dephosphorylated NFAT protein levels in control, SFRP2- treated endothelial cells, and SFRP2 and tacrolimus treated cells. After one hour of treatment of 2H11 endothelial cells with mouse recombinant SFRP2 (7 nM), nuclear NFATc3 was increased 2 fold (p = 0.003) (Fig. 5A). However, when tacrolimus (10 uM) was added to SFRP2 (7 nM) treatment, there was no increase in nuclear NFATc3 protein levels (Fig. 5A, Figure S2C). To evaluate whether the reduction in nuclear NFAT induced by tacrolimus is from nuclear translocation and not simply reduced expression of NFAT, we evaluated the effect of tacrolimus treatment on NFATc3 protein in whole cell lysates. There was no reduction Tacrolimus inhibits SFRP2 and VEGF -Induced Endothelial Cell Tube Formation We previously showed that tacrolimus can inhibit SFRP2- mediated tube formation in vitro [5]. In this study we explored whether tacrolimus would also inhibit VEGF induced tube formation, as VEGF has been reported to activate the calci- neurin/NFAT pathway [1–3]. 2H11 endothelial cells were stimulated with either SFRP2 or VEGF +/2 tacrolimus to determine if tacrolimus inhibited tube formation stimulated by both mitogens. Tacrolimus (1 mM) inhibited SFRP2 induced 2H11 tube formation by 71% (p = 0.005, Fig. 6A), and tube formation was inhibited in a concentration dependent manner. Tacrolimus (1 mM) also inhibited VEGF induced 2H11 tube formation by 67% (p = 0.004, Fig. 6B). Tacrolimus was not cytotoxic to 2H11 cells, as less than 5% of tacrolimus-treated cells took up trypan blue dye (data not shown). This shows that tacrolimus inhibits both SFRP2 and VEGF induced tube formation and therefore could potentially block angiogenesis stimulated by both mitogens. Tacrolimus inhibits breast tumor and endothelial cell migration The migration properties of tacrolimus on 2H11 endothelial cells and MMTV-neu breast carcinoma cells were evaluated using a scratch wound assay. Tacrolimus (1 uM) inhibited the migration of MMTV-neu cell migration at 24 hours by 45% (p = 0.04, Fig. 7A), and inhibited the migration of SFRP2 stimulated 2H11 cells at 20 hours by 20% (P = 0.008, Fig. 7B). This shows that tacrolimus has a direct effect on breast tumor cells in addition to its antiangiogenic effect. Discussion This study provides further evidence for a novel pathway of SFRP2 signaling in endothelial cells through activation of NFAT, a pathway shared with other angiogenesis stimulators such as Figure 6. Tacrolimus inhibited SFRP2 and VEGF stimulated endothelial tube formation in vitro. 2H11 endothelial cells were plated in Matrigel as described in ‘‘Material and Methods’’. A) SFRP2 induced endothelial tube formation after 6 hours, which was inhibited by tacrolimus in a concentration dependent manner. B) VEGF induced endothelial tube formation after 6 hours, which was inhibited by tacrolimus in a concentration dependent manner. doi:10.1371/journal.pone.0020412.g006 PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20412