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Title page for ETD etd-12102010-121704
|Type of Document
||Atwood, Allison Annette
||Regulation of C/EBPbeta1 in transformed mammary epithelial cells and the role of C/EBPbeta1 in oncogene-induced senescence
- breast cancer
|Date of Defense
Overexpression of activated oncogenes such as Ras(V12) in primary cells induces senescence rather than transformation, thus suppressing tumorigenesis. C/EBPbeta is required for Ras(V12)-induced senescence in mouse embryonic fibroblasts. C/EBPbeta is a transcription factor in which three protein isoforms exist due to alternative translation at three in-frame methionines. Upregulation of IL6 by C/EBPbeta is necessary for oncogene-induced senescence (OIS), but the specific isoform of C/EBPbeta has not been investigated. We show that the C/EBPbeta1 isoform upregulates IL6 and induces senescence when introduced into normal fibroblasts. In contrast to normal fibroblasts, overexpression of Ras(V12) in MCF10A cells, an immortalized mammary epithelial cell line, leads to transformation and degradation of C/EBPbeta1, suggesting a mechanism exists to bypass OIS in these cells. When MCF10A-Ras cells are forced to express C/EBPbeta1, these cells degrade p52-C/EBPbeta1. Treatment of MCF10A-Ras-C/EBPbeta1 cells with the cdk inhibitor roscovitine stabilizes p52-C/EBPbeta1. Cdk2 phosphorylates C/EBPbeta1 on Thr235. Mutation of Thr235 of alanine in C/EBPbeta1 restores stability of p52-C/EBPbeta1 expression in MCF10A-Ras cells, indicating that phosphorylation of C/EBPbeta1 on Thr235 by cdk2 is leading to degradation of p52-C/EBPbeta1 in the MCF10A-Ras cells.
C/EBPbeta1 is also regulated by Ras in breast cancer cells by the post-translational modification SUMO-2/3. Although p52-C/EBPbeta1 is not detected in anti-C/EBPbeta1 immunoblots of breast cancer cells, higher molecular weight bands are frequently observed. Exogenously expressed C/EBPbeta1 is sumoylated in breast cancer cells, and the higher molecular weight bands observed in anti-C/EBPbeta1 immunoblots of breast cancer cell lines is sumoylated C/EBPbeta1. Phosphorylation oftentimes enhances sumoylation, and phosphorylation cascades, including the Ras pathway, are activated in breast cancer cells. C/EBPbeta1 can be phosphorylated on Thr235 by Erk-2, a downstream effector of Ras. Phosphorylation of purified C/EBPbeta1 by Erk-2 enhances sumoylation, in vitro. Additionally, sumoylated C/EBPbeta1 is phosphorylated on Thr235 and the C/EBPbeta1T235A mutant shows a 3-fold decrease in sumoylation as compared to wild type C/EBPbeta1. Taken together, degradation or sumoylation of C/EBPbeta1 by Ras activation may represent mechanisms to bypass OIS.
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