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-, IFNß-, 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.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Ergosterol/analogs & derivatives , Intercellular Signaling Peptides and Proteins/metabolism , Lactones/pharmacology , Receptors, Cytokine/metabolism , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolism , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cytokine Receptor gp130/metabolism , DNA-Binding Proteins , Epidermal Growth Factor/metabolism , Ergosterol/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interferon-beta/metabolism , Interleukin-6/metabolism , Mice , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , NIH 3T3 Cells , Phosphorylation/drug effects , Platelet-Derived Growth Factor/metabolism , bcl-X Protein/metabolismABSTRACT
Mitotic catastrophe occurs when cells enter mitosis with damaged DNA or excess centrosomes. Cells overexpressing the centrosome protein CP110 or depleted of cyclin F, which targets CP110 for destruction, have more than two centrosomes and undergo mitotic catastrophe. Our studies show centrosome reduplication and mitotic catastrophe in osteosarcoma cells inducibly expressing a p27Kip1 mutant (termed p27K) that binds cyclins but not cyclin-dependent kinases (CDKs). p27K inhibited cell proliferation but not CDK activity or cell cycle progression. It did not induce apoptosis; however, cells expressing p27K had more than two centrosomes and, indicative of mitotic catastrophe, irregularly shaped nuclei or multiple micronuclei. p27K interacted with cyclin F in vivo (as did endogenous p27Kip1) and displaced cyclin F from CP110. Depletion of CP110 rescued p27K-expressing cells from centrosome reduplication and mitotic catastrophe. Collectively, our data show that p27Kip1 can perturb mitosis and suggest that it does so by sequestering cyclin F, which prevents its interaction with and the subsequent degradation of CP110, ultimately resulting in centrosome reduplication, mitotic catastrophe and abrogation of cell proliferation.