Mutagenic analysis of the role of the PSDTSSK domain of human SLUG protein in its repressor function in breast cancer cells

B89

SLUG is a transcriptional repressor protein implicated to have major roles in the oncogenesis and metastasis of human breast cells. It inhibits the expressions of many genes whose products are involved in the cell-cell adhesion and cellular proliferation. Our objective is to study the structure activity relationships in the SLUG protein in order to develop a mechanism based chemical intervention that abolishes the repressor function of SLUG. Towards that goal, we identified two essential domains in the repressor segment of the human SLUG (hSLUG) protein by deletion/modification analyses. They are the N-terminal SNAG domain and the internal PSDTSSK domain (P-domain). Deletion/alteration of each of these domains abrogated the repressor activity of the hSLUG protein. Repressor activity of the native hSLUG or the hSLUG derivatives were determined by the over expression of the protein in MDA-MB-468 cells (SLUG-negative cell) that are expressing Renilla luciferase from the human claudin 7 gene promoter. The human claudin 7 gene promoter has seven E2 box sequences which are potential binding sites for hSLUG. Thus, the activity of this promoter is very sensitive to hSLUG action and is an ideal assay system for the structure function analyses of hSLUG. Since the SNAG domain is common to several transcriptional repressors including SNAIL, Gfi1 etc., the development of aptamers against the SNAG domain will not only inhibit hSLUG but also other SNAG domain bearing proteins. We are thus focusing on the detailed analysis of the P-domain of hSLUG to find out whether a peptide aptamer against this domain may be a specific inhibitor against hSLUG protein function in human breast cells. One of the breast cancer disparities among African American and Caucasian women is that in the former population breast cancer is significantly more aggressive with a quick and fatal outcome. SLUG is supposedly a determinant factor for the aggressiveness of breast cancer. Development of a specific functional antagonist against SLUG may thus particularly help to slow the fatal development of breast carcinoma in African American women. Supported by the DOD-CDMRP IDEA Grant #W81XWH-06-1-0466, Komen Foundation for the Cure grant BCTR0707627 and the NCI SPORE subproject grant # 3 P50 CA098131 03S1 to GC.