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Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.

Zhang X, Blaskovich MA, Forinash KD, Sebti SM - Br. J. Cancer (2014)

Bottom Line: The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation.Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1.These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Department, Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.

ABSTRACT

Background: The binding of STAT3 and STAT5 to growth factor and cytokine receptors such as EGFR and IL-6 receptor gp130 is critical to their activation and ability to contribute to malignant transformation. Therefore, interfering with these biochemical processes could lead to the discovery of novel anticancer agents.

Methods: Co-immunoprecipitation, western blotting, microscopy, DNA binding, invasion, and soft agar assays as well as a mouse model were used to investigate the mechanism by which the natural product Withacnistin (Wit) inhibits STAT 3/5 tyrosine phosphoryaltion and activation.

Results: Wit blocks EGF- and IL-6-stimulated binding of STAT3 and STAT5 to EGFR and gp130. Wit inhibits EGF-, PDGF-, IL-6-, IFNβ-, and GM-CSF-stimulation of tyrosine phosphorylation of STAT3 and STAT5 but not of EGFR or PDGFR. The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation. Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1. Finally, Wit induces apoptosis, inhibits anchorage-dependent and -independent growth and invasion, and causes breast tumour regression in an ErbB2-driven transgenic mouse model.

Conclusions: These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

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Related in: MedlinePlus

Withacnistin inhibits EGF- and IL-6-stimulated EGFR and gp130 receptor recruitment and subsequent tyrosine phosphorylation of STAT3 and STAT5. (A) MDA-MB-468 cells were serum starved for 48 h and pretreated with Wit for the indicated times then stimulated with EGF at 100 ng ml−1 for 10 min. Cells were then harvested and run for western blot analysis as described under Materials and Methods. (B) MDA-MB-468 cells were serum starved for 24 h and pretreated with Wit at increasing concentrations for 5 min, and then stimulated with EGF at 100 ng ml−1 for 30 min. Cells were then harvested and run for co-immunoprecipitation analysis as described under Materials and Methods. (C) U266 and (D) RPMI 8226 cells were serum starved for 24 h and pretreated with Wit for 5 min, and then stimulated with IL-6 at 10 ng ml−1 for 30 min. S3I designates S3I-1757, a control STAT3–EGFR association inhibitor. ‘C' designates non-starved cells growing in 10% serum. The data in A–D are representative of three independent experiments each.
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fig1: Withacnistin inhibits EGF- and IL-6-stimulated EGFR and gp130 receptor recruitment and subsequent tyrosine phosphorylation of STAT3 and STAT5. (A) MDA-MB-468 cells were serum starved for 48 h and pretreated with Wit for the indicated times then stimulated with EGF at 100 ng ml−1 for 10 min. Cells were then harvested and run for western blot analysis as described under Materials and Methods. (B) MDA-MB-468 cells were serum starved for 24 h and pretreated with Wit at increasing concentrations for 5 min, and then stimulated with EGF at 100 ng ml−1 for 30 min. Cells were then harvested and run for co-immunoprecipitation analysis as described under Materials and Methods. (C) U266 and (D) RPMI 8226 cells were serum starved for 24 h and pretreated with Wit for 5 min, and then stimulated with IL-6 at 10 ng ml−1 for 30 min. S3I designates S3I-1757, a control STAT3–EGFR association inhibitor. ‘C' designates non-starved cells growing in 10% serum. The data in A–D are representative of three independent experiments each.

Mentions: Previously, we showed that Wit inhibits the constitutive levels of tyrosine-phosphorylated STAT3 in human tumours where STAT3 is hyperactive. However, the mechanism by which Wit reduces P-Tyr-STAT3 levels is not known. Furthermore, whether Wit also interferes with signalling of STAT5, another STAT protein involved in oncogenesis is not known. To address these important questions, first we determined whether Wit could interfere with the ability of growth factors such as EGF to stimulate the tyrosine phosphorylation of STAT3 and STAT5. To this end, we pretreated serum-starved MDA-MB-468 breast cancer cells with Wit for increasing lengths of time before EGF stimulation, and processed the cells for western blotting as described under Materials and Methods. Figure 1A shows that in the absence of Wit, EGF stimulated the tyrosine phosphorylation of both STAT3 and STAT5. Pretreatment with Wit for as little as 2–10 min inhibited EGF-stimulated STAT3 tyrosine phosphorylation partially, and complete inhibition occurred within 15–30 min. Figure 1A also shows that Wit pretreatment was able to inhibit EGF-stimulated STAT5 tyrosine phosphorylation but this effect was delayed relative to the inhibition of STAT3 phosphorylation with complete inhibition not occurring until 60–120 min.


Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.

Zhang X, Blaskovich MA, Forinash KD, Sebti SM - Br. J. Cancer (2014)

Withacnistin inhibits EGF- and IL-6-stimulated EGFR and gp130 receptor recruitment and subsequent tyrosine phosphorylation of STAT3 and STAT5. (A) MDA-MB-468 cells were serum starved for 48 h and pretreated with Wit for the indicated times then stimulated with EGF at 100 ng ml−1 for 10 min. Cells were then harvested and run for western blot analysis as described under Materials and Methods. (B) MDA-MB-468 cells were serum starved for 24 h and pretreated with Wit at increasing concentrations for 5 min, and then stimulated with EGF at 100 ng ml−1 for 30 min. Cells were then harvested and run for co-immunoprecipitation analysis as described under Materials and Methods. (C) U266 and (D) RPMI 8226 cells were serum starved for 24 h and pretreated with Wit for 5 min, and then stimulated with IL-6 at 10 ng ml−1 for 30 min. S3I designates S3I-1757, a control STAT3–EGFR association inhibitor. ‘C' designates non-starved cells growing in 10% serum. The data in A–D are representative of three independent experiments each.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150266&req=5

fig1: Withacnistin inhibits EGF- and IL-6-stimulated EGFR and gp130 receptor recruitment and subsequent tyrosine phosphorylation of STAT3 and STAT5. (A) MDA-MB-468 cells were serum starved for 48 h and pretreated with Wit for the indicated times then stimulated with EGF at 100 ng ml−1 for 10 min. Cells were then harvested and run for western blot analysis as described under Materials and Methods. (B) MDA-MB-468 cells were serum starved for 24 h and pretreated with Wit at increasing concentrations for 5 min, and then stimulated with EGF at 100 ng ml−1 for 30 min. Cells were then harvested and run for co-immunoprecipitation analysis as described under Materials and Methods. (C) U266 and (D) RPMI 8226 cells were serum starved for 24 h and pretreated with Wit for 5 min, and then stimulated with IL-6 at 10 ng ml−1 for 30 min. S3I designates S3I-1757, a control STAT3–EGFR association inhibitor. ‘C' designates non-starved cells growing in 10% serum. The data in A–D are representative of three independent experiments each.
Mentions: Previously, we showed that Wit inhibits the constitutive levels of tyrosine-phosphorylated STAT3 in human tumours where STAT3 is hyperactive. However, the mechanism by which Wit reduces P-Tyr-STAT3 levels is not known. Furthermore, whether Wit also interferes with signalling of STAT5, another STAT protein involved in oncogenesis is not known. To address these important questions, first we determined whether Wit could interfere with the ability of growth factors such as EGF to stimulate the tyrosine phosphorylation of STAT3 and STAT5. To this end, we pretreated serum-starved MDA-MB-468 breast cancer cells with Wit for increasing lengths of time before EGF stimulation, and processed the cells for western blotting as described under Materials and Methods. Figure 1A shows that in the absence of Wit, EGF stimulated the tyrosine phosphorylation of both STAT3 and STAT5. Pretreatment with Wit for as little as 2–10 min inhibited EGF-stimulated STAT3 tyrosine phosphorylation partially, and complete inhibition occurred within 15–30 min. Figure 1A also shows that Wit pretreatment was able to inhibit EGF-stimulated STAT5 tyrosine phosphorylation but this effect was delayed relative to the inhibition of STAT3 phosphorylation with complete inhibition not occurring until 60–120 min.

Bottom Line: The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation.Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1.These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Department, Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.

ABSTRACT

Background: The binding of STAT3 and STAT5 to growth factor and cytokine receptors such as EGFR and IL-6 receptor gp130 is critical to their activation and ability to contribute to malignant transformation. Therefore, interfering with these biochemical processes could lead to the discovery of novel anticancer agents.

Methods: Co-immunoprecipitation, western blotting, microscopy, DNA binding, invasion, and soft agar assays as well as a mouse model were used to investigate the mechanism by which the natural product Withacnistin (Wit) inhibits STAT 3/5 tyrosine phosphoryaltion and activation.

Results: Wit blocks EGF- and IL-6-stimulated binding of STAT3 and STAT5 to EGFR and gp130. Wit inhibits EGF-, PDGF-, IL-6-, IFNβ-, and GM-CSF-stimulation of tyrosine phosphorylation of STAT3 and STAT5 but not of EGFR or PDGFR. The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation. Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1. Finally, Wit induces apoptosis, inhibits anchorage-dependent and -independent growth and invasion, and causes breast tumour regression in an ErbB2-driven transgenic mouse model.

Conclusions: These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

Show MeSH
Related in: MedlinePlus