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The role of calcineurin/NFAT in SFRP2 induced angiogenesis--a rationale for breast cancer treatment with the calcineurin inhibitor tacrolimus.

Siamakpour-Reihani S, Caster J, Bandhu Nepal D, Courtwright A, Hilliard E, Usary J, Ketelsen D, Darr D, Shen XJ, Patterson C, Klauber-Demore N - PLoS ONE (2011)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
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 µM) 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.

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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.
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pone-0020412-g006: 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.

Mentions: 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 calcineurin/NFAT pathway [1]–[3]. 2H11 endothelial cells were stimulated with either SFRP2 or VEGF +/− tacrolimus to determine if tacrolimus inhibited tube formation stimulated by both mitogens. Tacrolimus (1 µM) 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 µM) 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.


The role of calcineurin/NFAT in SFRP2 induced angiogenesis--a rationale for breast cancer treatment with the calcineurin inhibitor tacrolimus.

Siamakpour-Reihani S, Caster J, Bandhu Nepal D, Courtwright A, Hilliard E, Usary J, Ketelsen D, Darr D, Shen XJ, Patterson C, Klauber-Demore N - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108822&req=5

pone-0020412-g006: 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.
Mentions: 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 calcineurin/NFAT pathway [1]–[3]. 2H11 endothelial cells were stimulated with either SFRP2 or VEGF +/− tacrolimus to determine if tacrolimus inhibited tube formation stimulated by both mitogens. Tacrolimus (1 µM) 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 µM) 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.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
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 µM) 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.

Show MeSH
Related in: MedlinePlus