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The major goal of this study was to investigate whether the estrogen-inducible transcription factor ZEB-1 contributed to the progression of breast carcinoma. For this study, we pursued and obtained sufficient numbers of staged, matched breast cancer biopsy tissues. I developed a novel protocol for multiplex real-time PCR so we could analyze the expression of four genes in one reaction simultaneously. The second year of work on these projects showed great progress and completion of our goals. Keeping in mind a new family member, ZEB-2, I designed and performed the real-time assays on a number of breast tissue samples. We found that the expression of both ZEB-1 and ZEB-2 are significantly increased in invasive breast cancers, compared to their normal match tissue, and ZEB-1 is no longer under the regulation of estrogen. To investigate the possibility of gene amplification, I created a ZEB-1 specific DNA program for quantitative Southern blot analysis and have successfully tested this probe on ovarian tissue biopsies. I have also constructed the ecdysone-inducible MCF-7 cell line that is needed for Task 4. In this line, ZEB-1 expression can be turned on or off with the presence or absence of ecdysone, and thus the ability of ZEB-1 to promote cell invasion can now be studied by Matrigel invasion assay. In support of our hypotheses, we have shown that ZEB-1 expression correlates with invasive breast cancer and that its expression is no longer regulated by estrogen.

The major goal of the proposed research is to test the hypothesis that the estrogen-inducible transcription factor AREB6/ZEB contributes to the progression of breast carcinoma. In specific, I am investigating whether the expression of AREB6/ZEB becomes deregulated from estrogen control and, if so, at what stage of carcinogenesis. To test this, staged breast cancer samples are being collected and assayed by real-time PCR for the level of AREB6/ZEB mRNA. Furthermore, I am assessing relative estrogen levels by determining the amounts of pS2 and progesterone receptor mRNA in the biopsies, also by real-time PCR. The use of real-time PCR and the measurement of pS2 and progesterone receptor mRNAs represent changes in strategy from the original proposal. These changes were made because we were informed that serum estrogen levels were an unreliable measure of tissue estrogen levels (especially in postmenopausal women), because we were having difficulty collecting paired biopsy samples and serum, and because a real-time PCR machine became available. In addition, we are also measuring the amount of another family member ZEB-2, which has been implicated recently in invasion. At this point, I have virtually all the samples and reagents in hand to complete the proposed studies in the upcoming year., The original document contains color images. All DTIC reproductions will be in black and white.