DeMore1,2,3* Abstract The Role of Calcineurin/NFAT in SFRP2 Induced Angiogenesis—A Rationale for Breast Cancer Treatment with the Calcineurin Inhibitor Tacrolimus Sharareh Siamakpour-Reihani1, Joseph Caster2, Desh Bandhu Nepal2, Andrew Courtwright2, Eleanor Hilliard3, Jerry Usary1, David Ketelsen 1 , David Darr1, Xiang Jun Shen1, Cam Patterson1,3, Nancy Klauber- 1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophilin FKBPB12. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, resulting in inhibition of nuclear translocation of nuclear factor of activated T-cells (NFAT). There is increasing data supporting a critical role of NFAT in mediating angiogenic responses stimulated by both vascular endothelial growth factor (VEGF) and a novel angiogenesis factor, secreted frizzled- related protein 2 (SFRP2). Since both VEGF and SFRP2 are expressed in breast carcinomas, we hypothesized that tacrolimus would inhibit breast carcinoma growth. Using IHC (IHC) with antibodies to FKBP12 on breast carcinomas we found that FKBP12 localizes to breast tumor vasculature. Treatment of MMTV-neu transgenic mice with tacrolimus (3 mg/kg i.p. daily) (n = 19) resulted in a 73% reduction in the growth rate for tacrolimus treated mice compared to control (n = 15), p = 0.003; which was associated with an 82% reduction in tumor microvascular density (p,0.001) by IHC. Tacrolimus (1 mM) inhibited SFRP2 induced endothelial tube formation by 71% (p = 0.005) and inhibited VEGF induced endothelial tube formation by 67% (p = 0.004). To show that NFATc3 is required for SFRP2 stimulated angiogenesis, NFATc3 was silenced with shRNA in endothelial cells. Sham transfected cells responded to SFRP2 stimulation in a tube formation assay with an increase in the number of branch points (p,0.003), however, cells transfected with shRNA to NFATc3 showed no increase in tube formation in response to SFRP2. This demonstrates that NFATc3 is required for SFRP2 induced tube formation, and tacrolimus inhibits angiogenesis in vitro and breast carcinoma growth in vivo. This provides a rationale for examining the therapeutic potential of tacrolimus at inhibiting breast carcinoma growth in humans. Citation: Siamakpour-Reihani S, Caster J, Bandhu Nepal D, Courtwright A, Hilliard E, et al. (2011) The Role of Calcineurin/NFAT in SFRP2 Induced Angiogenesis—A Rationale for Breast Cancer Treatment with the Calcineurin Inhibitor Tacrolimus. PLoS ONE 6(6): e20412. doi:10.1371/journal.pone.0020412 Editor: Eric J. Bernhard, National Cancer Institute, United States of America Received October 26, 2010; Accepted May 2, 2011; Published June 3, 2011 Copyright: 2011 Siamakpour-Reihani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funded by National Cancer Institute (1R01CA142657-01A1) and NIH National Cancer Institute Breast Cancer Specialized Program of Research Excellence grants P50-CA58223 to NK-D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Discovery of Novel Targets for Angiogenesis Inhibition, Provisional patent application no. 61/053,397. Inventors: Nancy Klauber-DeMore, MD, Cam Patterson, MD, PhD., Rajendra Bhati, MD filed 05/15/2008. Dr. Klauber-DeMore is co-founder and Chief Scientific Officer of Enci Therapeutics, Inc. Dr. Patterson is co-founder and Chief Executive Officer of Enci Therapeutics, Inc. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Enci Therapeutics had no role in funding this study and none of the authors has received a salary from the company. * E-mail: nancy_demore@med.unc.edu Introduction Tumor angiogenesis is regulated by multiple proangiogenic factors, of which the most widely studied is vascular endothelial growth factor (VEGF). One of the pathways through which VEGF stimulates angiogenesis is through activation of calcineurin/ nuclear factor of activated T-cells (NFAT) signaling [1–3]. Recent work has identified a novel angiogenesis factor, secreted frizzled- related protein 2 (SFRP2), which is expressed in the vasculature of a wide variety of tumors including human breast carcinoma [4,5]. SFRP2 stimulates angiogenesis in the mouse Matrigel plug assay, induces endothelial cell migration and tube formation in vitro, and protects against hypoxia-induced apoptosis in endothelial cells [5]. SFRP2 belongs to a large family of secreted frizzle-related proteins (SFRPs) which are related to the Wnt signaling cascade. Wnt proteins have been grouped into two classes – canonical and noncanonical – on the basis of their activity in cell lines or in vivo assays. The core of the canonical Wnt pathway is the stability of beta catenin [6]. SFRPs have been regarded as inhibitors of the canonical Wnt-beta catenin pathway [6], while recent studies have shown that SFRP2 can increase nuclear beta catenin levels [7–10]. In contrast we previously found that treatment of endothelial cells with SFRP2 (at angiogenic doses) resulted in no change in nuclear beta catenin levels in endothelial cells [5], suggesting that SFRP2 does not stimulate angiogenesis through inhibition or activation of the Wnt/beta catenin pathway. Noncanonical Wnts activate other signaling pathways, such as the Wnt/Ca2+ pathway [11]. The Wnt/Ca2+ pathway is a beta catenin-independent pathway for which signaling is mediated through transient increases in cytoplasmic free calcium which PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20412