Berna Uygur

Date of Award

5-2013

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Wen Shu Wu

Second Committee Member

Lucy Liaw

Third Committee Member

Ah Kau Ng

Abstract

Slug is a transcription factor of the Snail/Slug zinc-finger family and is implicated in metastasis and invasion of tumor cells. Mechanism by which Slug promotes migration, invasion and cell proliferation of prostate cancers is unclear.

To investigate the role of Slug in prostate cancer, I examined Slug expression in mouse prostate tumors, human prostate tumors, and human prostate cancer cell lines LNCaP, PC-3, DU145 and 22RV1. Slug expression was found elevated at transcriptional and protein level in prostate cancer cells. I also demonstrated that forced expression of Slug inhibited proliferation of prostate cancer cells PC-3 and DU-145. Conversely, reduced expression of Slug by shRNA promoted growth of PC-3 cancer cells. Consistent with these data, I found that forced expression of Slug in prostate cancer cells led to G1 cell- cycle arrest.

Furthermore, ectopic expression of Slug decreased cyclin D1 expression in both PC-3 and DU-145 cells, and knockdown of Slug by shRNA upregulated cyclin D1 expression in these cancer cells. In addition, I demonstrated that ectopic expression of cyclin D1 relieved Slug-mediated inhibition of proliferation of prostate cancer cells.

The role of Slug in metastasis and invasion of prostate tumor cells was examined. It was demonstrated that forced expression of Slug elevated CXCR4 and CXCL12 expression in human prostate cancer cell lines PC-3, DU145, 22RV1, and LNCaP; conversely, reduced expression of Slug by shRNA down regulated CXCR4 and CXCL12 expression at RNA and protein levels in prostate cancer cells. Furthermore, ectopic expression of Slug increased MMP9 expression and activity in PC-3, 22RV1, and DU-145 cells, and Slug knockdown by shRNA downregulated MMP9 expression. I showed that CXCL12 is required for Slug-mediated MMP9 expression in prostate cancer cells. Moreover, I found that migration and invasion of prostate cancer cells was increased by ectopic expression of Slug and decreased by Slug knockdown. Notably, knockdown of CXCL12 by shRNA impaired Slug-mediated migration and invasion in prostate cancer cells. My data suggest that CXCL12 and Slug regulate migration and invasion of prostate cancer cells independent of cell growth. I provided the first compelling evidence that upregulation of autocrine CXCL12 is a major mechanism underlying Slug-mediated migration and invasion of prostate cancer cells. Our findings suggest that CXCL12 is a therapeutic target for prostate cancer metastasis.

In addition to the role of Slug in proliferation, metastasis, and invasion of prostate cancer cells, molecular mechanism of drug resistance promoted by Slug was investigated. It was shown that reduced expression of Slug by shRNA up regulated PTEN expression in human prostate cancer cell lines, PC-3, LNCaP cells. Conversely, elevated PTEN expression due to Slug knockdown caused loss of phosphorylated AKT (pAKT) expression in prostate cancer cell lines, PC-3, LNCaP cells. I have showed that elevated endogenous level Slug expression caused loss of PTEN expression and conversely up regulation of pAKT expression in human prostate cancer tissue samples. Reduced expression of both PTEN and Slug by shRNA mediated higher drug resistance and higher cancer stem cell population in PC-3 and LNCaP cells.

I provided the first compelling evidence that Slug is a negative regulator of PTEN expression. Our findings in this study indicated that Slug promotes drug resistance and cancer stem cell phenotype generation through PTEN regulation and I might suggest that Slug mediated prostate cancer cell metastasis and cancer cell drug resistance is required PTEN down regulation. Slug and PTEN might be prognostic marker and potential therapeutic target for prostate cancer.

In summary, Slug plays multiple roles in prostate cancer development. It reduces cell proliferation by promoting cyclin D1 degradation, increases cell motility by upregulation of CXCR4/CXCL12 axis in prostate cancer cells. Lastly, Slug increases drug resistance through cancer stem cell population by down regulation of PTEN.

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