ERBB2 gene is overexpressed in about thirty percent of most aggressive human breast cancers. The overexpression results from gene amplification and deregulated transcription. We1-3 and others4 have identified the involvement of AP-2 family transcription factors in ERBB2 gene overexpression in breast cancer cells. The 6kb fragment of the ERBB2 promoter contains several AP-2 binding sites. The binding of the transcription factors to the proximal and the distal sites of the endogenous promoter was shown by Chromatin Immunoprecipitation2.

In most cell lines, high concentrations of AP-2 are transcriptionaly inactive. In contrast, breast cancer cell lines overexpressing ERBB2 contain high amounts of transcriptionaly active AP-2 proteins. We hypothesize that these cells contain specific cofactors responsible for maintaining AP-2 activity. Our goal is to identify these cofactors.

We have described YY1 protein as one of the factors contributing to AP-2 activity. Indeed, YY1 stimulates AP-2 transcriptional activity on the ERBB2 promoter. Moreover, YY1 binding to the endogenous ERBB2 promoter in breast cancer cells was detected by Chromatin Immunoprecipitation5.

Currently we are using siRNAs to knockdown AP-2 and YY1 expression in order to confirm the involvement of these factors in ERBB2 gene overexpression in breast cancer cells. In order to identify new AP-2 interacting factors in breast cancer cells we used GST pull-down technique. GST-tagged AP-2 was fixed on glutathione magnetic beads and incubated with nuclear proteins from BT-474 cells. The attached proteins were eluted and analyzed on 2D gels. The spots were cut-out and the proteins were identified by mass spectrometry. Ku70 and Ku80 were identified as AP-2 interacting proteins by this method. To find out if Ku proteins modulate AP-2 activity on ERBB2 promoter, Ku expression vectors were co- transfected with reporter vectors containing ERBB2 promoter fragments. Preliminary results show cooperation between Ku80 and AP-2 on ERBB2 promoter. Moreover, addition of YY1 expression vector further increased the transcriptional activity. We plan to assess by Chromatin Immunoprecipitation and siRNA the involvement of Ku on ERBB2 gene expression.

Reference List

1. Grooteclaes,M. et al. (1999) Cancer Res. 59, 2527-2531

2. Delacroix,L. et al. (2005) DNA Cell Biol. 24, 582-594

3. Vernimmen,D. et al. (2003) Biochem J 370, 323-9

4. Bosher,J.M. et al. (1996) Oncogene 13, 1701-7

5. Begon,D.Y. et al. (2005) J. Biol. Chem. 280, 24428-24434

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA