Graduation Year

2008

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Cancer Biology

Major Professor

Hong-Gang Wang, Ph.D.

Keywords

Anoikis, Bif-1, Cancer, Ku70, Mcl-1

Abstract

Apoptosis is an inherent suicide mechanism that cells invoke for a variety of reasons including embryo cavitation, tissue homeostasis, excessive DNA damage and aberrant oncogene activation. Apoptosis is regulated by a diverse set of proteins including, but not limited to, the Bcl-2 family. This family set is comprised of both pro-death and pro-survival proteins whose relative expression, localization and/or modifications regulate the balance between life and death for each cell. The keystones to this system are the proapoptotic proteins Bax and Bak, which are regulated by their conformation and localization. However, the exact mechanisms by which Bax and Bak become activated remains to be resolved. Similarly, research focusing on the cancer cell's ability to deregulate apoptosis by preventing the activation of Bax or Bak will provide further insight into the development of targeted therapies for cancer that will hopefully contribute to the cure of this formidable disease.

Src, the classic oncogenic kinase, has been found to deregulate Bax activation in response to the detachment of a cell from its substratum support thereby preventing anoikis, the Bax-dependent apoptotic response involved in the impairment of metastatic dissemination of cancer. Our findings indicate that Src deregulates this response by altering the relative expression of Bcl-2 family members Mcl-1 and Bim through the PI3-K/Akt and Erk1/2 pathways. However, Src retains its ability to prevent anoikis even in the setting of Akt and Erk1/2 signaling inhibition. Further evaluation of the role of Src in this process revealed that Bif-1, a protein known to associate with and activate Bax, could be directly phosphorylated by Src which prevented the association of Bax with Bif-1 and impaired the anoikis response.

In addition, our studies have also found that Bax activation in response to treatment with type I and II histone deacetylase inhibitors is dependent on the expression of the tumor suppressor p53. Acetylation of p53 at carboxy-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here, we demonstrate that p53 acetylation at K320/K373/K382 is also required for its transcription-independent functions in Bax activation, ROS production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) SAHA and LAQ824. Knockout of p53 in HCT116 cells markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cancer cells to HDACi-mediated Bax-dependent apoptosis, whereas knockdown of endogenous mutant p53 inhibited HDACi-induced apoptosis. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70.

The association between these two proteins was acetylation independent, but acetylation of p53 could prevent and disrupt the Ku70/Bax complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

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