Figure 7. Tacrolimus inhibited breast cancer and endothelial cell migration in a scratch wound migration assay. A) MMTV-neu breast cancer cells were plated in a scratch wound assay as described in ‘‘Material and Methods’’. A wound was formed with a 1 mm pipette tip, and tacrolimus or control was added to the wells. Migration was measured at various time points with an ocular micrometer. Tacrolimus statistically significantly inhibited MMTV-neu breast tumor migration at 1 and 10 mM (p,0.05). B) 2H11 cells were plated in a tube formation assay without SFRP2 (Control 1.5% DMSO), with SFRP2 7 nM and 1.5% DMSO, or with SFRP2 (7 nM)+tacrolimus (0.1–10 mM in 1.5% DMSO). SFRP2 induced endothelial cell migration compared to control, which was statistically significantly inhibited by tacrolimus at 0.1, 1 and 10 mM. doi:10.1371/journal.pone.0020412.g007 VEGF. Although SFRPs have been regarded as inhibitors of the canonical Wnt-beta catenin pathway [6], four recent studies have shown that SFRP2 can act as a Wnt agonist rather than an antagonist. SFRP2 has been found to increase nuclear beta catenin levels in: 1) cardiomyocytes exposed to hypoxia and treated with mouse recombinant SFRP2 [7], 2) MCF7 cells with stable transfectants of SFRP2 [8], 3) mammary canine tumors with increased expression of SFRP2 [9], and hypoxic adipose tissue derived stem cells [10]. In contrast, we previously found that there was no change in nuclear beta catenin in endothelial cells stimulated with SFRP2 at doses that induced angiogenesis in vitro, although we did see an increase in nuclear beta catenin at a four-fold higher dose. In this study we have now confirmed with siRNA silencing that beta catenin is not required for SFRP2 induced endothelial tube formation. We also previously found that NFATc3 is activated in endothelial cells after SFRP2 treatment, and now confirm with shRNA silencing that NFATc3 is required for SFRP2 induced tube formation. This suggests that targeting NFAT with calcineurin inhibitors is a potential therapeutic approach to inhibit angiogenesis induced by both VEGF and SFRP2. Tacrolimus Inhibits Breast Cancer Growth In Vivo Several lines of evidence suggest that calcineurin inhibitors like tacrolimus might have efficacy against tumor growth. Tacrolimus has previously been shown to inhibit the growth of an NFAT overexpressing mouse leukemia xenograft in vivo [22] and the SVR angiosarcoma xenograft in vivo [5].Tacrolimus has not been tested for its ability to inhibit breast cancer in humans, however in a large series of patients receiving tacrolimus following liver transplanta- tion the de novo incidence of breast cancer was found to be 1.9 times lower than matched controls from the SEER database [23]. This correlation has only been observed in specific patient populations but in light of our study demonstrating that the binding partner for tacrolimus, FKBP12, is expressed in the vasculature of human breast tumors, and that tacrolimus inhibits the growth rate of MMTV-neu transgenic breast carcinomas in mice, there is good rationale for prospectively investigating the clinical utility of tacrolimus in breast cancer treatment. There are several calcineurin inhibitors that are FDA approved for the prevention of organ transplant rejection, although only tacrolimus and cyclosporine inhibit NFAT. Sirolimus (rapomycin) and everolimus (RAD001, a derivative of rapomycin) have the same intracellular target as tacrolimus, FKBP12, but unlike tacrolimus these drugs inhibit mTOR and have no effect on NFAT [24]. Because the safety profile of tacrolimus is better than cyclosporine, we chose to study tacrolimus for efficacy at inhibiting breast tumor growth. The anticancer potential of calcineurin inhibitors like tacrolimus is likely not restricted to their ability to antagonize angiogenesis in the vasculature. Calcineurin/NFAT signaling cascades are important for proliferation and migration in a number of cell types. In this study we demonstrated that tacrolimus inhibited not only endothelial cell migration, but also breast tumor cell migration. Based on the critical importance of these modalities in cancer progression and maintenance it is not surprising that a growing number of studies have identified significant dysregulation of NFAT/calcineurin signaling in a variety of human tumors [25]. Increased NFAT/calcineurin activity has been observed in several human carcinomas including pancreatic [26], colon [27], and breast cell carcinoma [28]. In melanoma BRAF has been shown to activate NFAT to direct transcription of cyclooxygenase, a process which is also antagonized by tacrolimus [5]. Buchholz and colleagues have shown that about 70% of pancreatic carcinomas have elevated levels of nuclear NFATc1 compared to healthy pancreatic tissues. Using human-derived pancreatic carcinoma cell lines, they demonstrated that the nuclear localization of transcrip- tionally active NFATc1 is a calcineurin-dependent process that was inhibited by cyclosporine A. Treatment with cyclosporine A also inhibited in vitro cell cycle progression and anchorage- independent proliferation of the Panc1 cell line [26]. In vitro tacrolimus has been shown to inhibit hepatocellular carcinoma [29,30], and prostate cancer proliferation [31]. Studies such as these further support the investigation of calcineurin inhibitors like tacrolimus in anti-cancer regimens. In summary, we demonstrated that tacrolimus is able to inhibit in vitro tube formation stimulated by both SFRP2 and VEGF and inhibit the migration of endothelial and breast cancer cells. Tacrolimus may be particularly useful in the treatment of breast cancer as it attenuated breast tumor xenograft growth in vivo, and FKBP12 is expressed in the vasculature of human breast carcinomas. Given the utility of other antiangiogenic factors in breast cancer [32] and the previous observation of reduced breast cancer incidence following tacrolimus administration [23], there is ample rationale for examining the therapeutic potential of tacrolimus as part of an anti-breast cancer chemotherapeutic regimen. PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20412