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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 STAT3 transcriptional activity and expression of downstream target genes. (A) MDA-MB-468 cells were transiently transfected with pLucSRE or pLucTKS3 along with β-gal reporter genes and were then treated with vehicle or Wit as indicated. The cytosolic extracts were prepared and analysed for luciferase activity as described in Materials and Methods. C and M designate control and mock transfection, respectively. (B) MDA-MB-468 cells were treated for 24 h with the indicated concentrations of Wit and processed for western blotting with the indicated antibodies as described in Materials and Methods. S3I designates S3I-1757, a control STAT3 inhibitor. The data in A and B are representative of three and two independent experiments, respectively.
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fig4: Withacnistin inhibits STAT3 transcriptional activity and expression of downstream target genes. (A) MDA-MB-468 cells were transiently transfected with pLucSRE or pLucTKS3 along with β-gal reporter genes and were then treated with vehicle or Wit as indicated. The cytosolic extracts were prepared and analysed for luciferase activity as described in Materials and Methods. C and M designate control and mock transfection, respectively. (B) MDA-MB-468 cells were treated for 24 h with the indicated concentrations of Wit and processed for western blotting with the indicated antibodies as described in Materials and Methods. S3I designates S3I-1757, a control STAT3 inhibitor. The data in A and B are representative of three and two independent experiments, respectively.

Mentions: We next evaluated the ability of Wit to inhibit STAT3-dependent transcriptional activation using luciferase reporter assays. To this end, MDA-MB-468 cells were transiently co-transfected with a STAT3-responsive promoter-firefly luciferase reporter (pLucTKS3) and β-gal reporter used to normalise the transfection efficiency. To determine the selectivity of Wit to suppress STAT3-dependent over STAT3-independent transcriptional activation, MDA-MB-468 cells were also co-transfected with SRE promoter-renilla luciferase reporter (pLucSRE) and β-gal reporter. Figure 4A shows that compared with mock transfected cells, cells transfected with the STAT3-responsive reporter (pLucTKS3) had increased luciferase activity in the absence of drug treatment. Less luciferase activity was observed when the cells were treated with Wit. Wit inhibited STAT3-dependent but not STAT3-independent transcriptional activity as demonstrated by the minimal effect it had on SRE-driven luciferase activity (Figure 4A).


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 STAT3 transcriptional activity and expression of downstream target genes. (A) MDA-MB-468 cells were transiently transfected with pLucSRE or pLucTKS3 along with β-gal reporter genes and were then treated with vehicle or Wit as indicated. The cytosolic extracts were prepared and analysed for luciferase activity as described in Materials and Methods. C and M designate control and mock transfection, respectively. (B) MDA-MB-468 cells were treated for 24 h with the indicated concentrations of Wit and processed for western blotting with the indicated antibodies as described in Materials and Methods. S3I designates S3I-1757, a control STAT3 inhibitor. The data in A and B are representative of three and two independent experiments, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Withacnistin inhibits STAT3 transcriptional activity and expression of downstream target genes. (A) MDA-MB-468 cells were transiently transfected with pLucSRE or pLucTKS3 along with β-gal reporter genes and were then treated with vehicle or Wit as indicated. The cytosolic extracts were prepared and analysed for luciferase activity as described in Materials and Methods. C and M designate control and mock transfection, respectively. (B) MDA-MB-468 cells were treated for 24 h with the indicated concentrations of Wit and processed for western blotting with the indicated antibodies as described in Materials and Methods. S3I designates S3I-1757, a control STAT3 inhibitor. The data in A and B are representative of three and two independent experiments, respectively.
Mentions: We next evaluated the ability of Wit to inhibit STAT3-dependent transcriptional activation using luciferase reporter assays. To this end, MDA-MB-468 cells were transiently co-transfected with a STAT3-responsive promoter-firefly luciferase reporter (pLucTKS3) and β-gal reporter used to normalise the transfection efficiency. To determine the selectivity of Wit to suppress STAT3-dependent over STAT3-independent transcriptional activation, MDA-MB-468 cells were also co-transfected with SRE promoter-renilla luciferase reporter (pLucSRE) and β-gal reporter. Figure 4A shows that compared with mock transfected cells, cells transfected with the STAT3-responsive reporter (pLucTKS3) had increased luciferase activity in the absence of drug treatment. Less luciferase activity was observed when the cells were treated with Wit. Wit inhibited STAT3-dependent but not STAT3-independent transcriptional activity as demonstrated by the minimal effect it had on SRE-driven luciferase activity (Figure 4A).

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