Date of Award

Winter 2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Timothy J. Bos

Committee Member

Ann E. Campbell

Committee Member

Julie A. Kerry

Committee Member

Kenneth D. Somers

Abstract

Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotypic changes related to malignant progression including a shift to estrogen independent growth, increased cell motility and invasion. Concurrent with these phenotypic changes are alterations to cellular gene expression patterns. One gene that becomes highly upregulated is SPARC (secreted protein acidic and rich in cysteine). Increased SPARC expression is associated with malignant progression in a variety of different cancers, although little is known regarding the mechanisms of SPARC gene regulation. Therefore, the objectives of this study were: (1) to determine the mechanisms by which c-Jun regulates SPARC gene expression, and (2) to determine the contribution of SPARC to c-Jun induced phenotype in a MCF7 breast cancer model system.

In order to determine the role of SPARC in c-Jun mediated oncogenic progression, we over-expressed SPARC in MCF7 cells and blocked its expression in the c-Jun/MCF7 cell line. We found that antisense mediated suppression of SPARC dramatically inhibits both cell motility and invasion in this c-Jun/MCF7 model. In contrast, stable overexpression of SPARC in the parental MCF7 cell line was not sufficient to stimulate cell motility or invasion suggesting that SPARC cooperates with other c-Jun target genes to establish a pro-invasive phentoytpe.

In order to determine the mechanism(s) of c-Jun induced SPARC gene activation, we started by analyzing DNA binding and transactivation using the human SPARC promoter. The activity of the full-length SPARC promoter (-1409/+28) was 15-30 fold higher in c-Jun over-expressing cells compared to vector control cells. Promoter deletion analysis revealed that a region between -120 and -70 conferred c-Jun responsiveness. This region does not contain an AP-1 binding site, but does contain a GC rich element which is recognized in vitro and in vivo by Sp1. Importantly, chromatin immunoprecipitation analysis demonstrated that c-Jun is physically associated with the SPARC proximal promoter region during gene activation.

Further analysis of the SPARC promoter sequence, including the c-Jun responsive region, revealed the presence of multiple CpG sequences. Methylation of cytosine residues in a CpG context has been shown to inhibit gene expression. Therefore, we examined the contribution of DNA methylation to SPARC gene regulation. Analysis of MCF7 cells, in which SPARC expression is undetectable, revealed methylation of the SPARC promoter at both distal and proximal sites.

DOI

10.25777/mwe1-9996

ISBN

9780542407109

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