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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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Beta catenin was not required for SFRP2 induced endothelial tube formation: 2H11 endothelial cells were transfected with siRNA to beta catenin or sham transfected.A) Western blot results of siRNA to beta catenin showed silencing of beta catenin by 80%. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) 2H11 endothelial cell tube formation assay. Cells were plated in Matrigel as described in “Material and Methods”. After 6 hours the number of branch points were counted. There was no significant decrease in de novo or SFRP2 (7 nm) induced tube formation following the silencing of beta catenin. Full-length blots/gels are presented in Figure S2A.
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pone-0020412-g003: Beta catenin was not required for SFRP2 induced endothelial tube formation: 2H11 endothelial cells were transfected with siRNA to beta catenin or sham transfected.A) Western blot results of siRNA to beta catenin showed silencing of beta catenin by 80%. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) 2H11 endothelial cell tube formation assay. Cells were plated in Matrigel as described in “Material and Methods”. After 6 hours the number of branch points were counted. There was no significant decrease in de novo or SFRP2 (7 nm) induced tube formation following the silencing of beta catenin. Full-length blots/gels are presented in Figure S2A.

Mentions: To show whether beta catenin is involved in SFRP2-stimulated angiogenesis, beta catenin was silenced in 2H11 endothelial cells with siRNA and their ability to undergo tube formation was compared to sham-transfected cells. Western blot analyses of 2H11 cells transfected with siRNA to beta catenin demonstrated 80% knockdown of beta catenin (Fig. 3A, Figure S2). Some studies have shown that SFRP2 is an inhibitor of beta catenin [6]; if the mechanism through which SFRP2 stimulated tube formation is through inhibition of beta catenin signaling, then we would expect that silencing beta catenin in endothelial cells would increase tube formation. However, we found no difference in de novo tube formation between sham transfected cells and siRNA beta catenin transfected cells (Fig. 3B). Other studies have shown that SFRP2 can increase nuclear beta catenin levels [7]–[10]; if the mechanism through which SFRP2 stimulated tube formation was through activation of beta catenin signaling, then we would expect that silencing beta catenin in endothelial cells would block SFRP2 induced tube formation. However, there was no difference in tube formation between sham-transfected cells stimulated with SFRP2 (7 nM) and siRNA beta catenin transfected cells stimulated with SFRP2 (7 nM) [Fig. 3B]. This demonstrates that beta catenin is not required for SFRP2 induced 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)

Beta catenin was not required for SFRP2 induced endothelial tube formation: 2H11 endothelial cells were transfected with siRNA to beta catenin or sham transfected.A) Western blot results of siRNA to beta catenin showed silencing of beta catenin by 80%. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) 2H11 endothelial cell tube formation assay. Cells were plated in Matrigel as described in “Material and Methods”. After 6 hours the number of branch points were counted. There was no significant decrease in de novo or SFRP2 (7 nm) induced tube formation following the silencing of beta catenin. Full-length blots/gels are presented in Figure S2A.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020412-g003: Beta catenin was not required for SFRP2 induced endothelial tube formation: 2H11 endothelial cells were transfected with siRNA to beta catenin or sham transfected.A) Western blot results of siRNA to beta catenin showed silencing of beta catenin by 80%. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) 2H11 endothelial cell tube formation assay. Cells were plated in Matrigel as described in “Material and Methods”. After 6 hours the number of branch points were counted. There was no significant decrease in de novo or SFRP2 (7 nm) induced tube formation following the silencing of beta catenin. Full-length blots/gels are presented in Figure S2A.
Mentions: To show whether beta catenin is involved in SFRP2-stimulated angiogenesis, beta catenin was silenced in 2H11 endothelial cells with siRNA and their ability to undergo tube formation was compared to sham-transfected cells. Western blot analyses of 2H11 cells transfected with siRNA to beta catenin demonstrated 80% knockdown of beta catenin (Fig. 3A, Figure S2). Some studies have shown that SFRP2 is an inhibitor of beta catenin [6]; if the mechanism through which SFRP2 stimulated tube formation is through inhibition of beta catenin signaling, then we would expect that silencing beta catenin in endothelial cells would increase tube formation. However, we found no difference in de novo tube formation between sham transfected cells and siRNA beta catenin transfected cells (Fig. 3B). Other studies have shown that SFRP2 can increase nuclear beta catenin levels [7]–[10]; if the mechanism through which SFRP2 stimulated tube formation was through activation of beta catenin signaling, then we would expect that silencing beta catenin in endothelial cells would block SFRP2 induced tube formation. However, there was no difference in tube formation between sham-transfected cells stimulated with SFRP2 (7 nM) and siRNA beta catenin transfected cells stimulated with SFRP2 (7 nM) [Fig. 3B]. This demonstrates that beta catenin is not required for SFRP2 induced 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