The Calcineurin Inhibitor Tacrolimus Inhibits Breast Tumor Growth In Vivo.

Background: We recently reported a novel angiogenesis factor, secreted frizzle-related protein 2 (SFRP2), that stimulates angiogenesis via a calcineurin/ NFAT pathway and is highly expressed in human breast tumor endothelium (Courtwight et. al., Cancer Research 2009). NFAT is a transcription factor that mediates vascular development and VEGF and bFGF-induced angiogenesis. Tacrolimus (FK506) is a calcineurin inhibitor that inhibits NFAT activation and is FDA approved to prevent organ transplant rejection. Based on its mechanism of action, we hypothesized that tacrolimus would inhibit angiogenesis and breast tumor growth. Materials and Methods: Tumor growth in vivo: MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p. for 21 days or control when tumors became palpable. Tumor volumes were measured with 3D ultrasound. Endothelial tube formation in vitro: ECMatrix was solidified into wells of a 96 well plate and endothelial cells were seeded onto the matrix at a concentration of 1x10 4 cells/well with SFRP2 7nM or VEGFA 60ng/ml. Tacrolimus 1µM or 1.5% DMSO control was added to the wells. The number of branch points were counted after 8 hours. MMTV-neu migration in vitro: Breast tumor cells were plated at 10,000 cells/well into a 96 well plate and allowed to become confluent. The cells were quiesced for 18 hours and tacrolimus (1µM) or 0.5% DMSO control was added to the wells and the wound was formed using a 1 ml pipette tip. Migration distance was measured with an ocular micrometer after 16 hours. Western blot analyses: Endothelial cells were treated for 1 hour with negative control, SFRP2 7nM, or SFRP2 7nM plus tacrolimus 10 µM. Nuclear protein was extracted and Western blot for NFATc3 was performed. Statistics: Means were compared with a two-tailed t-test. Results: Tumor growth in vivo: Tacrolimus inhibited the growth rate of MMTV-neu tumors by 49% (n=12 treated, n=9 controls, p=0.04), without signs of toxicity. Endothelial tube formation: Tacrolimus (1µM) inhibited SFRP2 induced endothelial tube formation by 64% (0.002, n=4), and VEGF induced tube formation by 69% (p=0.004, n=4). MMTV-neu tumor cell migration: Tacrolimus (1µM) inhibited MMTV-neu cell migration by 36% (p Western blot for NFATc3: Treatment of endothelial cells with SFRP2 induced nuclear NFATc3 at 1 hour compared to control, and this increase was abolished by treatment with tacrolimus. Conclusion: The calcineurin inhibitor tacrolimus inhibits the growth rate of MMTV-neu transgenic tumors in vivo . The in vitro cellular effects of tacrolimus include inhibiting SFRP2 and VEGF-induced endothelial tube formation, abolishing SFRP2 activation of NFATc3 in endothelial cells, and inhibiting MMTV-neu cell migration, demonstrating a direct effect of tacrolimus on both the endothelial and tumor cell compartments. This establishes that NFAT may be a favorable therapeutic target to inhibit angiogenesis and breast cancer growth. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3170.