| Activation of the Ras pathway leads to phosphorylation of the transcription factor Ets-2, which results in activation of target genes including; the growth factor HB-EGF, extracellular proteases urokinase plasminogen activator (uPA) and MMP3, and the apoptotic regulator bcl-xL.; In breast cancer cells, we have defined two new potential targets for Ets-2 action, the tumor suppressor BRCA1 and the metalloproteinase MT1-MMP. Analysis of both the BRCA1 and MT1-MMP promoters indicates potential binding sites for the Ets family of transcription factors. In addition, expression of MT1-MMP mRNA is high in the invasive cell line MDA-MB-231 that expresses phosphorylated Ets-2, and low in the non-invasive cell line MCF7 that does not express phosphorylated Ets-2. BRCA1 mRNA is expressed in the opposite fashion, that is high in MCF7 and low in MDA231. Analysis of the MT1-MMP promoter in these cells indicated that the MT1-MMP promoter is 5–10 fold more active in MDA231 compared to MCF7. Co-expression of Ets-2 increased MT1-MMP promoter activity in MCF7 cells 5–10 fold. Analysis of the BRCA1 promoter by transient transfection demonstrated that the BRCA1 promoter is approximately 10 times more active in MCF7 cells compared to MDA231 cells. Co-expression of Ets-2 lowered BRCA1 promoter activity in MCF7 cells approximately 10 fold.; To further study Ets-2 action, we constructed MCF7 cell lines that conditionally express Ets-2. Cell lines expressing low levels of Ets-2 appeared to have a normal epithelial morphology and grew normally, however, cell lines expressing high levels of unphosphorylated Ets-2 had a more mesenchymal appearance and ceased to proliferate. Preliminary studies suggest that high levels of unphosphorylated Ets-2 in MCF7 cells leads to apoptosis. In addition, upon induction of Ets-2 expression, BRCA1 mRNA expression levels decreased, whereas, both uPA and MMP3 expression levels increased.; In summary, we have shown that Ets-2 not only acts as a transcriptional activator of uPA, MMP3, and MT1-MMP, genes that promote metastasis, but may also act as a transcriptional repressor to inhibit expression of the tumor suppressor gene BRCA1. The role of Ets-2 in the development and progression of mammary carcinogenesis has been further defined as a result of our studies. |
