Limits...
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.

Show MeSH

Related in: MedlinePlus

Tacrolimus inhibited the growth rate of MMTV-neu transgenic mouse tumors.MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p., no treatment control, 20% intralipid i.p. control beginning when tumors became palpable, and was continued for 21 days. Tumor volumes were measured on day 7, 14 and 21, and the growth rate (percent change in tumor volume per day) was compared between control (no treatment), control (20% intralipid) and tacrolimus treated groups. There was no statistically significant difference between control (untreated) and control (20% intralipid) groups, and therefore the controls were combined for analyses. At 21 days there was a 73% reduction in the growth rate for tacrolimus treated mice compared to no treatment (n = 19 tacrolimus treated, n = 15 control, *p = 0.003). Tacrolimus treated mice were significantly different from control (no treatment, p = 0.01, and control 20% intralipid, p = 0.01). There was no weight loss or lethargy in the tacrolimus-treated mice or the 20% intralipid treated mice.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3108822&req=5

pone-0020412-g001: Tacrolimus inhibited the growth rate of MMTV-neu transgenic mouse tumors.MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p., no treatment control, 20% intralipid i.p. control beginning when tumors became palpable, and was continued for 21 days. Tumor volumes were measured on day 7, 14 and 21, and the growth rate (percent change in tumor volume per day) was compared between control (no treatment), control (20% intralipid) and tacrolimus treated groups. There was no statistically significant difference between control (untreated) and control (20% intralipid) groups, and therefore the controls were combined for analyses. At 21 days there was a 73% reduction in the growth rate for tacrolimus treated mice compared to no treatment (n = 19 tacrolimus treated, n = 15 control, *p = 0.003). Tacrolimus treated mice were significantly different from control (no treatment, p = 0.01, and control 20% intralipid, p = 0.01). There was no weight loss or lethargy in the tacrolimus-treated mice or the 20% intralipid treated mice.

Mentions: To evaluate whether tacrolimus inhibits breast carcinoma growth, we compared MMTV-neu transgenic mice treated with tacrolimus in 20% intralipid (n = 19) at 3 mg/kg/day i.p. for 21 days to control mice that received no treatment (n = 9) or control with 20% intralipid i.p. (n = 6). There was no statistically significant difference in the growth rate between control with no treatment (227±51) and control with 20% intralipid (279±101, p = 0.6), demonstrating that the control vehicle did not affect tumor growth. Therefore the control groups were combined for analyses. The mean growth rate (% change in tumor volume) after 21 days was 68±25 in tacrolimus treated mice, compared to the combined controls (n = 15) 248±48, which is a reduction of 73% (p = 0.003, Fig. 1). When tacrolimus is compared to the no treatment controls the p = 0.01, and to the 20% intralipid controls the p = 0.01. There were no signs of toxicity (i.e., diarrhea, infection, lethargy, or weight loss) after treatment. This demonstrates that tacrolimus inhibits the growth rate of breast carcinoma in vivo.


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 the growth rate of MMTV-neu transgenic mouse tumors.MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p., no treatment control, 20% intralipid i.p. control beginning when tumors became palpable, and was continued for 21 days. Tumor volumes were measured on day 7, 14 and 21, and the growth rate (percent change in tumor volume per day) was compared between control (no treatment), control (20% intralipid) and tacrolimus treated groups. There was no statistically significant difference between control (untreated) and control (20% intralipid) groups, and therefore the controls were combined for analyses. At 21 days there was a 73% reduction in the growth rate for tacrolimus treated mice compared to no treatment (n = 19 tacrolimus treated, n = 15 control, *p = 0.003). Tacrolimus treated mice were significantly different from control (no treatment, p = 0.01, and control 20% intralipid, p = 0.01). There was no weight loss or lethargy in the tacrolimus-treated mice or the 20% intralipid treated mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020412-g001: Tacrolimus inhibited the growth rate of MMTV-neu transgenic mouse tumors.MMTV-neu transgenic mice were treated with tacrolimus 3 mg/kg/day i.p., no treatment control, 20% intralipid i.p. control beginning when tumors became palpable, and was continued for 21 days. Tumor volumes were measured on day 7, 14 and 21, and the growth rate (percent change in tumor volume per day) was compared between control (no treatment), control (20% intralipid) and tacrolimus treated groups. There was no statistically significant difference between control (untreated) and control (20% intralipid) groups, and therefore the controls were combined for analyses. At 21 days there was a 73% reduction in the growth rate for tacrolimus treated mice compared to no treatment (n = 19 tacrolimus treated, n = 15 control, *p = 0.003). Tacrolimus treated mice were significantly different from control (no treatment, p = 0.01, and control 20% intralipid, p = 0.01). There was no weight loss or lethargy in the tacrolimus-treated mice or the 20% intralipid treated mice.
Mentions: To evaluate whether tacrolimus inhibits breast carcinoma growth, we compared MMTV-neu transgenic mice treated with tacrolimus in 20% intralipid (n = 19) at 3 mg/kg/day i.p. for 21 days to control mice that received no treatment (n = 9) or control with 20% intralipid i.p. (n = 6). There was no statistically significant difference in the growth rate between control with no treatment (227±51) and control with 20% intralipid (279±101, p = 0.6), demonstrating that the control vehicle did not affect tumor growth. Therefore the control groups were combined for analyses. The mean growth rate (% change in tumor volume) after 21 days was 68±25 in tacrolimus treated mice, compared to the combined controls (n = 15) 248±48, which is a reduction of 73% (p = 0.003, Fig. 1). When tacrolimus is compared to the no treatment controls the p = 0.01, and to the 20% intralipid controls the p = 0.01. There were no signs of toxicity (i.e., diarrhea, infection, lethargy, or weight loss) after treatment. This demonstrates that tacrolimus inhibits the growth rate of breast carcinoma in vivo.

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