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Publication Date

2009-12-21

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2009-11-12

First Committee Member

Karoline Briegel

Second Committee Member

Terace Fletcher

Third Committee Member

Kerry Burnstein

Fourth Committee Member

Zafar Nawaz

Fifth Committee Member

Francesco DeMayo

Abstract

Steroid receptor coactivators modulate the final outcome of hormone induced gene transcription by steroid receptors. E6-associated protein (E6-AP), an E3 ubiquitin ligase, acts as a coactivator of steroid receptors, including estrogen receptor (ER). In this study, we elucidated the contribution of E6-AP to ER-dependent gene transcription in breast cancer cells. siRNA-mediated knockdown of E6-AP abrogates transcription of classic ER target genes, GREB1 and pS2, suggesting that E6-AP is essential for normal transactivation function of ER. In order to understand the global influence of E6-AP in ER-dependent gene transcription, we used gene expression microarrays under E6-AP knockdown conditions to identify ER target genes which are regulated by E6-AP. Our microarray analysis revealed 455 genes which are differentially regulated by E6-AP. Pathway analysis revealed that E6-AP regulated genes were involved in cell cycle. Cell cycle profiling at various time points of estrogen treatment reveals that under E6-AP knockdown conditions, breast cancer cells progress slowly through S phase and eventually fail to proliferate. Knockdown of E6-AP has no effect on ovarian and uterine cells, suggesting that E6-AP has cell specific roles. Our analysis suggests that knockdown of E6-AP reduces the levels of early (C-Myc and Cyclin-D1), mid (E2F1, E2F2 and E2F7) and late (BUB1, BUBR1, MAD2, NDC80, NUF2 and CASC5) estrogen-dependent cell cycle genes. Overall our data indicate that E6-AP is a major regulator of cell cycle in breast cancer cells. E6-AP also acts as a coactivator for androgen receptor (AR) and we studied the role of E6-AP in prostate gland development. We report the generation of transgenic mice which specifically over expresses E6-AP in the prostate gland. Prostate glands in these mice are larger when compared with its wild-type litter mates, corroborating our observations that knockout of E6-AP in mice leads to impaired prostate gland development. E6-AP transgenic mice also develop prostatic intra epithelial neoplasia after 18 months of age. In addition to these observations, we also show that over expression of E6-AP in the prostate gland leads to increased Akt signaling. In order to understand the mechanism by which E6-AP regulates prostate gland growth, we generated LNCaP cells that stably overexpress E6-AP protein. Data from these cell lines show that the levels of phosphatidylinositol 3-kinase, total Akt, phosphorylated Akt (active Akt) and its down-stream target protein, GSKβ are elevated, suggesting that E6-AP regulates the PI3K-Akt signaling pathway. We further show that E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased proliferation. Overall our data suggests that E6-AP regulates the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and tumorigenesis.

Keywords

Breast Cancer; MCF-7; Akt; Cell Cycle; Androgen Receptor; E6-AP; Estrogen Receptor

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