GSBS Dissertations and Theses

Approval Date

5-21-2010

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, MD/PhD Program, Cancer Biology and Biochemistry and Molecular Pharmacology

Subjects

Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53; Proto-Oncogene Proteins c-mdm2; HIV; C-terminal binding protein; Transaminases; Dissertations, UMMS

Abstract

Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer.

The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy.

In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies.

ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.

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