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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 decreased SFRP2 induced nuclear NFATc3 in 2H11 endothelial cells.A) 2H11 cells were treated with control (1.5% DMSO), mouse recombinant SFRP2 (7 nM)+1.5% DMSO; or SFRP2 7(nM)+tacrolimus 10 µM in 1.5% DMSO for 1 hour, and nuclear protein lysates were collected and analyzed by Western blot analyses probing for NFATc3 as described in “Material and Methods”. The loading control was TATA binding protein TBP antibodies (a nuclear marker). SFRP2 increased nuclear NFATc3 compared to control cells (p = 0.003). Full-length blots/gels are presented in Figure S2C. B) The experiment was repeated as above except that whole cell lysates rather than nuclear lysates were extracted and analyzed by Western blot analyses probing for NFATc3. The loading control was beta-tubulin. Tacrolimus did not inhibit total NFATc3 protein. Taken together this shows that tacrolimus inhibits SFRP2 induced NFATc3 nuclear translocation but not total protein levels.
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pone-0020412-g005: Tacrolimus decreased SFRP2 induced nuclear NFATc3 in 2H11 endothelial cells.A) 2H11 cells were treated with control (1.5% DMSO), mouse recombinant SFRP2 (7 nM)+1.5% DMSO; or SFRP2 7(nM)+tacrolimus 10 µM in 1.5% DMSO for 1 hour, and nuclear protein lysates were collected and analyzed by Western blot analyses probing for NFATc3 as described in “Material and Methods”. The loading control was TATA binding protein TBP antibodies (a nuclear marker). SFRP2 increased nuclear NFATc3 compared to control cells (p = 0.003). Full-length blots/gels are presented in Figure S2C. B) The experiment was repeated as above except that whole cell lysates rather than nuclear lysates were extracted and analyzed by Western blot analyses probing for NFATc3. The loading control was beta-tubulin. Tacrolimus did not inhibit total NFATc3 protein. Taken together this shows that tacrolimus inhibits SFRP2 induced NFATc3 nuclear translocation but not total protein levels.

Mentions: To evaluate the role of tacrolimus on the non-canonical Wnt/Ca++ pathway in SFRP2 induced angiogenesis, we compared nuclear dephosphorylated NFAT protein levels in control, SFRP2-treated endothelial cells, and SFRP2 and tacrolimus treated cells. After one hour of treatment of 2H11 endothelial cells with mouse recombinant SFRP2 (7 nM), nuclear NFATc3 was increased 2 fold (p = 0.003) (Fig. 5A). However, when tacrolimus (10 uM) was added to SFRP2 (7 nM) treatment, there was no increase in nuclear NFATc3 protein levels (Fig. 5A, Figure S2C). To evaluate whether the reduction in nuclear NFAT induced by tacrolimus is from nuclear translocation and not simply reduced expression of NFAT, we evaluated the effect of tacrolimus treatment on NFATc3 protein in whole cell lysates. There was no reduction of NFATc3 with tacrolimus treatment (Figure 5B). This shows that tacrolimus inhibits SFRP2 induced NFATc3 translocation in endothelial cells without reducing total NFATc3 protein.


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 decreased SFRP2 induced nuclear NFATc3 in 2H11 endothelial cells.A) 2H11 cells were treated with control (1.5% DMSO), mouse recombinant SFRP2 (7 nM)+1.5% DMSO; or SFRP2 7(nM)+tacrolimus 10 µM in 1.5% DMSO for 1 hour, and nuclear protein lysates were collected and analyzed by Western blot analyses probing for NFATc3 as described in “Material and Methods”. The loading control was TATA binding protein TBP antibodies (a nuclear marker). SFRP2 increased nuclear NFATc3 compared to control cells (p = 0.003). Full-length blots/gels are presented in Figure S2C. B) The experiment was repeated as above except that whole cell lysates rather than nuclear lysates were extracted and analyzed by Western blot analyses probing for NFATc3. The loading control was beta-tubulin. Tacrolimus did not inhibit total NFATc3 protein. Taken together this shows that tacrolimus inhibits SFRP2 induced NFATc3 nuclear translocation but not total protein levels.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020412-g005: Tacrolimus decreased SFRP2 induced nuclear NFATc3 in 2H11 endothelial cells.A) 2H11 cells were treated with control (1.5% DMSO), mouse recombinant SFRP2 (7 nM)+1.5% DMSO; or SFRP2 7(nM)+tacrolimus 10 µM in 1.5% DMSO for 1 hour, and nuclear protein lysates were collected and analyzed by Western blot analyses probing for NFATc3 as described in “Material and Methods”. The loading control was TATA binding protein TBP antibodies (a nuclear marker). SFRP2 increased nuclear NFATc3 compared to control cells (p = 0.003). Full-length blots/gels are presented in Figure S2C. B) The experiment was repeated as above except that whole cell lysates rather than nuclear lysates were extracted and analyzed by Western blot analyses probing for NFATc3. The loading control was beta-tubulin. Tacrolimus did not inhibit total NFATc3 protein. Taken together this shows that tacrolimus inhibits SFRP2 induced NFATc3 nuclear translocation but not total protein levels.
Mentions: To evaluate the role of tacrolimus on the non-canonical Wnt/Ca++ pathway in SFRP2 induced angiogenesis, we compared nuclear dephosphorylated NFAT protein levels in control, SFRP2-treated endothelial cells, and SFRP2 and tacrolimus treated cells. After one hour of treatment of 2H11 endothelial cells with mouse recombinant SFRP2 (7 nM), nuclear NFATc3 was increased 2 fold (p = 0.003) (Fig. 5A). However, when tacrolimus (10 uM) was added to SFRP2 (7 nM) treatment, there was no increase in nuclear NFATc3 protein levels (Fig. 5A, Figure S2C). To evaluate whether the reduction in nuclear NFAT induced by tacrolimus is from nuclear translocation and not simply reduced expression of NFAT, we evaluated the effect of tacrolimus treatment on NFATc3 protein in whole cell lysates. There was no reduction of NFATc3 with tacrolimus treatment (Figure 5B). This shows that tacrolimus inhibits SFRP2 induced NFATc3 translocation in endothelial cells without reducing total NFATc3 protein.

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