Defense Date

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Swati Palit Deb

Abstract

MDM2, the human homologue of the Mouse Double Minute 2 gene product, has been shown to be over-expressed in many cancers and to induce tumorigenesis. The role of MDM2 in oncogenesis was thought to be p53 dependent. However recent years have shown MDM2 to be a key player in a complex network of interactions that affect cell cycle, apoptosis, and tumorigenesis in a p53 independent manner. Here we report a novel p53 independent role for the multidimensional protein MDM2; its ability to induce phosphorylation of Akt at serine 473 residue. Transient and stable over-expression of MDM2 in cultured cell lines induces Akt phosphorylation. Silencing of MDM2 expression in cancer cells that over express MDM2 inhibits Akt phosphorylation suggesting endogenous MDM2 participates in Akt phosphorylation. Stable up-regulation of MDM2 expression reduced sensitivity of cells to chemotherapeutic drugs such as Etoposide, Carboplatin or Paclitaxel. The domain of MDM2 responsible for drug resistance overlaps with the Akt phosphorylation domain. An inhibitor of Akt phosphorylation abrogated MDM2-mediated Akt phosphorylation and reduction of Etoposide sensitivity indicating that MDM2 reduces Etoposide sensitivity of cancer cells by activating the Akt phosphorylation. A PI-3 kinase inhibitor Wortmannin inhibits the ability of MDM2 to induce Akt phosphorylation and silencing of Rictor, a known kinase of Akt, does not hamper the ability of MDM2 to induce phosphorylation of Akt. MDM2-mediated Akt phosphorylation does not require p53, and the p53-interaction domain of MDM2 is dispensable for Akt phosphorylation. The presence of MDM2 enhances the Insulin like Growth Factor 1 mediated activation of Akt. Further cells harboring MDM2 show enhanced Interleukin 8( IL8) activation, which could be a possible mechanism of Akt activation. Downstream of Akt activation we show increased events that have been correlated with Akt activation like increased Bcl-2 levels increased processing of NF-κB2, and GSK3α/β phosphorylation among others. Our observation reveals a novel signaling function of MDM2 important for regulation of cell growth and chemotherapeutic sensitivity through Akt phosphorylation.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2008

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