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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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Withacnistin inhibits P-STAT3 nuclear translocation and STAT3–DNA-binding activity. (A) MDA-MB-468 cells were cultured on cover slides overnight, serum starved for 24 h, pretreated with Wit at the indicated concentration for 2 h, and then stimulated with EGF at 100 ng ml−1 for 30 min. The cells were fixed using methanol for immunofluorescence analysis as described in Materials and Methods. (B) MDA-MB-468 cells were treated with either vehicle or Wit for the indicated time and the nuclear extracts were isolated from the treated cells as described in Materials and Methods. The nuclear extracts were then incubated with a biotin-labelled STAT3–DNA binding probe and the complexes were isolated using a STAT3–DNA-binding assay as described under Materials and Methods. The data in A and B are representative of two and three independent experiments.
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fig3: Withacnistin inhibits P-STAT3 nuclear translocation and STAT3–DNA-binding activity. (A) MDA-MB-468 cells were cultured on cover slides overnight, serum starved for 24 h, pretreated with Wit at the indicated concentration for 2 h, and then stimulated with EGF at 100 ng ml−1 for 30 min. The cells were fixed using methanol for immunofluorescence analysis as described in Materials and Methods. (B) MDA-MB-468 cells were treated with either vehicle or Wit for the indicated time and the nuclear extracts were isolated from the treated cells as described in Materials and Methods. The nuclear extracts were then incubated with a biotin-labelled STAT3–DNA binding probe and the complexes were isolated using a STAT3–DNA-binding assay as described under Materials and Methods. The data in A and B are representative of two and three independent experiments.

Mentions: The translocation of phosphorylated STAT3 from cytosol to the nucleus is required for STAT3 to regulate its target genes. The fact that Wit was able to inhibit receptor–STAT3 interaction (Figure 1) suggests that it would also inhibit nuclear accumulation of tyrosine-phosphorylated STAT3. To investigate this possibility, starved MDA-MB-468 cells were pretreated for 5 min with Wit before stimulation with EGF for 30 min. The cells were then fixed and subjected to immunofluorescence staining using a specific p-Tyr-705-STAT3 primary antibody and AlexaFluor 594 secondary antibody in mounting medium containing DAPI to stain the nuclei as described under Materials and Methods. Figure 3A shows that in starved cells some tyrosine-phosphorylated STAT3 is present. In the absence of Wit, EGF stimulation resulted in a robust increase in tyrosine-phosphorylated STAT3, which was localised predominantly in the nucleus. In contrast, the levels of P-STAT3 in the nucleus were decreased in Wit-treated cells in a concentration-dependent manner, indicating that Wit inhibited EGF-stimulated P-STAT3 nuclear translocation. The control STAT3 inhibitor S3I-1757 (Zhang et al, 2013) potently inhibited EGF-stimulated P-STAT3 nuclear translocation.


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 P-STAT3 nuclear translocation and STAT3–DNA-binding activity. (A) MDA-MB-468 cells were cultured on cover slides overnight, serum starved for 24 h, pretreated with Wit at the indicated concentration for 2 h, and then stimulated with EGF at 100 ng ml−1 for 30 min. The cells were fixed using methanol for immunofluorescence analysis as described in Materials and Methods. (B) MDA-MB-468 cells were treated with either vehicle or Wit for the indicated time and the nuclear extracts were isolated from the treated cells as described in Materials and Methods. The nuclear extracts were then incubated with a biotin-labelled STAT3–DNA binding probe and the complexes were isolated using a STAT3–DNA-binding assay as described under Materials and Methods. The data in A and B are representative of two and three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Withacnistin inhibits P-STAT3 nuclear translocation and STAT3–DNA-binding activity. (A) MDA-MB-468 cells were cultured on cover slides overnight, serum starved for 24 h, pretreated with Wit at the indicated concentration for 2 h, and then stimulated with EGF at 100 ng ml−1 for 30 min. The cells were fixed using methanol for immunofluorescence analysis as described in Materials and Methods. (B) MDA-MB-468 cells were treated with either vehicle or Wit for the indicated time and the nuclear extracts were isolated from the treated cells as described in Materials and Methods. The nuclear extracts were then incubated with a biotin-labelled STAT3–DNA binding probe and the complexes were isolated using a STAT3–DNA-binding assay as described under Materials and Methods. The data in A and B are representative of two and three independent experiments.
Mentions: The translocation of phosphorylated STAT3 from cytosol to the nucleus is required for STAT3 to regulate its target genes. The fact that Wit was able to inhibit receptor–STAT3 interaction (Figure 1) suggests that it would also inhibit nuclear accumulation of tyrosine-phosphorylated STAT3. To investigate this possibility, starved MDA-MB-468 cells were pretreated for 5 min with Wit before stimulation with EGF for 30 min. The cells were then fixed and subjected to immunofluorescence staining using a specific p-Tyr-705-STAT3 primary antibody and AlexaFluor 594 secondary antibody in mounting medium containing DAPI to stain the nuclei as described under Materials and Methods. Figure 3A shows that in starved cells some tyrosine-phosphorylated STAT3 is present. In the absence of Wit, EGF stimulation resulted in a robust increase in tyrosine-phosphorylated STAT3, which was localised predominantly in the nucleus. In contrast, the levels of P-STAT3 in the nucleus were decreased in Wit-treated cells in a concentration-dependent manner, indicating that Wit inhibited EGF-stimulated P-STAT3 nuclear translocation. The control STAT3 inhibitor S3I-1757 (Zhang et al, 2013) potently inhibited EGF-stimulated P-STAT3 nuclear translocation.

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