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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Defective p53 post-translational modification required for wild type p53 inactivation in malignant epithelial cells
Chad D Knights1, Yuangang Liu, Ettore Appella
1Department of Dermatology, Oregon Health & Science University, Portland, Oregon 97239, USA.
Abstract:
Mdm2 gene amplification occurs in benign and chemotherapy-responsive malignant tumors with wtp53 genes as well as in breast and epithelial cancers. Mdm2 amplification in benign tumors suggests that it is not sufficient for p53 inactivation in cancer, implying that other defects in the p53 pathway are required for malignancy. We investigated mechanisms of wtp53 protein inactivation in malignant conversion of epithelial cells by comparing clonally related initiated cells with their derivative cancerous cells that have mdm2 amplification. Deficiencies in p53 accumulation and activities in response to DNA damage were not due simply to Mdm2 destabilization of p53 protein, but to continued association of DNA-bound p53 with Mdm2 protein and lack of binding and acetylation by p300 protein. The aberrant interactions were not because of mdm2 amplification alone, because DNA-bound p53 protein from initiated cells failed to bind ectopically expressed Mdm2 or endogenous overexpressed Mdm2 from cancerous cells. Phosphorylations of endogenous p53 at Ser18, -23, or -37 were insufficient to dissociate Mdm2, because each was induced by UV in cancerous cells. Interestingly, phospho-mimic p53-T21E did dissociate the Mdm2 protein from DNA-bound p53 and recovered p300 binding and p21 induction in the cancerous cells. Thus wtp53 in malignant cells with mdm2 amplification can be inactivated by continued association of DNA-bound p53 protein with Mdm2 and failure of p300 binding and acetylation, coupled with a defect in p53 phosphorylation at Thr21. These findings suggest therapeutic strategies that address both p53/Mdm2 interaction and associated p53 protein defects in human tumors that have amplified mdm2 genes.
Insights
Mdm2 amplification alone doesn't cause cancer. Malignant conversion involves p53 protein inactivation through persistent Mdm2 binding, impaired p300 acetylation, and defective p53 phosphorylation at Thr21, suggesting new therapeutic targets.
Area of Science:
- Molecular Biology
- Cancer Research
- Cellular Signaling
Background:
- Mdm2 gene amplification is observed in various tumors, including breast and epithelial cancers, and also in benign tumors.
- Mdm2 amplification alone is insufficient for p53 pathway inactivation, suggesting additional defects are necessary for malignant transformation.
Purpose of the Study:
- To investigate the mechanisms of wild-type p53 (wtp53) protein inactivation during the malignant conversion of epithelial cells.
- To compare wtp53 inactivation mechanisms in initiated cells versus their malignant derivatives with Mdm2 amplification.
Main Methods:
- Comparative analysis of clonally related initiated and cancerous epithelial cells with Mdm2 amplification.
- Assessment of p53 protein accumulation, DNA binding, Mdm2 association, p300 binding, and acetylation.
- Investigation of p53 phosphorylation status at specific sites (Ser18, -23, -37, Thr21) in response to DNA damage (UV).
Main Results:
- p53 inactivation in malignant cells with Mdm2 amplification is due to sustained Mdm2 binding to DNA-bound p53 and impaired p300 binding/acetylation, not just Mdm2 destabilization.
- Aberrant p53-Mdm2 interactions persist even with Mdm2 overexpression, indicating complex regulatory defects.
- UV-induced p53 phosphorylation at Ser18, -23, or -37 did not dissociate Mdm2 in cancerous cells.
- A phospho-mimic mutation at p53 Thr21 (p53-T21E) successfully dissociated Mdm2, restored p300 binding, and induced p21 expression.
Conclusions:
- wtp53 inactivation in Mdm2-amplified malignant cells involves persistent Mdm2 association with DNA-bound p53, failure of p300 binding/acetylation, and a defect in p53 phosphorylation at Thr21.
- These findings highlight the complex interplay of Mdm2, p53, and p300 in tumorigenesis.
- Targeting both p53/Mdm2 interactions and specific p53 protein defects presents potential therapeutic strategies for Mdm2-amplified human tumors.
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