Graduation Year

2008

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Pathology and Cell Biology

Major Professor

Patricia A. Kruk, Ph.D.

Committee Member

Jin Q. Cheng, M.D., Ph.D.

Committee Member

Santo V. Nicosia, M.D.

Committee Member

Alvaro Monteiro, Ph.D.

Committee Member

Rebecca Sutphen, M.D.

Keywords

Ovarian cancer, Chemotherapy, Programmed cell death, Akt, Caspase 3

Abstract

Ovarian cancer is a deadly disease that kills an estimated 15,000 women annually in the United States. It is estimated that approximately 10% of ovarian cancers are due to familial inheritance. The most commonly mutated genes in familial ovarian cancer are BRCA1 and BRCA2. It has been reported that cells carrying the BRCA1 185delAG mutation undergo an enhanced caspase-3 mediated apoptotic response. Here, we report on the transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer cells. Cells transfected with the BRCA1 185delAG truncation protein (BRAt) showed increased levels of active caspase 3, increased cleavage of caspase 3 substrates, PARP and DFF45, and decreased XIAP and cIAP1 following staurosporine (STS) treatment. BRAt also reduced Akt phosphorylation and over expression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild type cells. Further, BRAt expression increased chemosensitivity in platinum resistant ovarian cancer cells. Similarly, maspin protein has been shown to sensitize breast carcinoma cells to STS-induced apoptosis. We provide the first evidence that BRAt is sufficient to induce maspin protein in IOSE cells. IOSE cell lines carrying the BRCA1 185delAG mutation showed higher maspin levels than wild-type BRCA1 IOSE cell lines. BRCA1 wild-type IOSE cells were transfected with BRAt protein and showed increased maspin mRNA levels and increased nuclear maspin protein levels as compared to control cells. Additionally, both heterozygous carriers of the BRCA1 185delAG mutation and cells transfected with BRAt protein show an increased ability to activate the maspin promoter as compared to control cells. The transcription factor AP1 is at least partially required for full activation of the maspin promoter in BRAt cells, as siRNA directed towards c-jun decreased activation of the full-length maspin promoter. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis by increasing nuclear maspin expression, suggesting a possible mechanism by which ovarian cancer patients with germline BRCA1 mutations may respond better to chemotherapy.

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