Down-regulation of promoter I.3 activity of the human aromatase gene in breast tissue by zinc-finger protein, snail (SnaH)

Aromatase (estrogen synthetase) is expressed in breast cancer tissue, and i n situ expression of the enzyme stimulates breast cancer growth. Promoter I .3 is one of the major promoters that control the expression of aromatase i n breast cancer tissue. Using the yeast one-hybrid approach to screen a hum an breast tissue hybrid cDNA expression library, we found that the zinc-fin ger transcriptional factor Snail (SnaH) interacted with a regulatory region near promoter I.3 of the human aromatase gene. DNA mobility shift assays a nd mutation analyses using recombinant SnaH protein expressed in Escherichi a coli have revealed that this protein interacts with a segment, 5'-CTGATGA AGT-3', which is between 66 and 76 bp upstream from the transcriptional sta rt site of promoter I.3, Using mammalian cell transfection experiments, Sna H was found to act as a repressor of promoter I.3 activity. Site-directed m utagenesis experiments have revealed that the NH2-terminal SNAG domain is i mportant for the repressor activity of SnaH, To demonstrate the inhibitory activity against aromatase expression, a stable SnaH-expressing MDA-MB-231 breast cancer cell line was generated, and the aromatase RNA messages in th e SnaH-transfected cell line were found to be 30% of those in the vector-tr ansfected cell line. Reverse transcription-PCR analysis on RNAs isolated fr om 12 cell lines has confirmed that SnaH is expressed at a higher level in normal breast epithelial cell and stromal fibroblast cell lines than in bre ast cancer cell lines. In addition, SnaH mRNA was detected in only 16 of 55 breast cancer specimens. On the other hand, aromatase mRNA was detected in 54 of the 55 specimens, Our results indicate that SnaH acts as a repressor that down-regulates the expression of aromatase in normal breast tissue by suppressing the function of promoter I.3. A reduction of the expression of SnaH in breast cancer tissue further suggests a cancer-protective role for this protein in normal breast tissue.