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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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NFATc3 was required for SFRP2 stimulated tube formation.A) ShRNA to SFRP2 in 2H11 endothelial cells showed 69% reduction in NFATc3 level by Western blot. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) Sham transfected 2H11 cells increased tube formation in response to SFRP2 (7 nM) (n = 3 for all groups, p<0.01), which was not seen in shRNA to NFATc3 transfected cells. Full-length blots/gels are presented in Supplemental Figure S2B. Pictures of sham transfected cells and shRNA to NFATc3 transfected cells (both stimulated with SFRP2 (7 nM)) are in Figure S3.
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pone-0020412-g004: NFATc3 was required for SFRP2 stimulated tube formation.A) ShRNA to SFRP2 in 2H11 endothelial cells showed 69% reduction in NFATc3 level by Western blot. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) Sham transfected 2H11 cells increased tube formation in response to SFRP2 (7 nM) (n = 3 for all groups, p<0.01), which was not seen in shRNA to NFATc3 transfected cells. Full-length blots/gels are presented in Supplemental Figure S2B. Pictures of sham transfected cells and shRNA to NFATc3 transfected cells (both stimulated with SFRP2 (7 nM)) are in Figure S3.

Mentions: 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.


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)

NFATc3 was required for SFRP2 stimulated tube formation.A) ShRNA to SFRP2 in 2H11 endothelial cells showed 69% reduction in NFATc3 level by Western blot. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) Sham transfected 2H11 cells increased tube formation in response to SFRP2 (7 nM) (n = 3 for all groups, p<0.01), which was not seen in shRNA to NFATc3 transfected cells. Full-length blots/gels are presented in Supplemental Figure S2B. Pictures of sham transfected cells and shRNA to NFATc3 transfected cells (both stimulated with SFRP2 (7 nM)) are in Figure S3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020412-g004: NFATc3 was required for SFRP2 stimulated tube formation.A) ShRNA to SFRP2 in 2H11 endothelial cells showed 69% reduction in NFATc3 level by Western blot. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) Sham transfected 2H11 cells increased tube formation in response to SFRP2 (7 nM) (n = 3 for all groups, p<0.01), which was not seen in shRNA to NFATc3 transfected cells. Full-length blots/gels are presented in Supplemental Figure S2B. Pictures of sham transfected cells and shRNA to NFATc3 transfected cells (both stimulated with SFRP2 (7 nM)) are in Figure S3.
Mentions: 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.

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