The GATA-3 transcription factor is a critical determinant of mammary cell fate. GATA-3 expression is high in normal human breast epithelium and in estrogen receptor positive tumors, as GATA-3 directly regulates expression of estrogen receptor alpha (ER?). Mutations in the GATA-3 gene are found in a subset of human breast cancers;induced re-expression of GATA-3 in tumor cell lines leads to decreased proliferation and an increase in differentiation markers. Recent studies demonstrate that GATA-3 inhibits and limits the potential for metastasis to the lung. GATA-3 is also important in T cell development and differentiation, in which Fliz-1, the fetal liver zinc finger protein, directly represses transcription by binding to sequences within the first intron of the GATA-3 gene;however the potential for Fliz1 to regulate GATA3 in mammary cells is completely unknown. HYPOTHESIS: Fliz1 binds the GATA3 intronic regulatory region in mammary cells to repress transcription. Decreased Fliz1 expression will lead to increased GATA-3 and ER?. Increased GATA-3 will cause decreased tumor cell invasiveness, and increased hormone sensitivity.
SPECIFIC AIMS : The proposed studies will (1) Determine if Fliz1 binds the GATA3 gene and regulates GATA-3 and hence ER? expression in mammary cell lines. Expression vectors driving a fusion of Fliz1 and green fluorescent protein (Fliz1-GFP) will be introduced into mammary cells. Chromatin immunoprecipitation using anti-GFP antibodies will determine if the fusion product binds the GATA3 intronic regulatory region in mouse and human breast tumor cells. GATA3 and ER? expression will be tested in normal mouse mammary cells and mouse and human breast cancer cells overexpressing Fliz1 and those in which Fliz1 is silenced via RNAi. (2) Assess the effect of Fliz1 expression on the ability of mammary tumor cells to proliferate and invade in in vitro assays. The ability of genetically manipulated cells to proliferate and invade basement membrane in response to estradiol will be tested. The role of Fliz1 in normal and induced apoptotic processes will be assessed. (3) Determine if Fliz-1 silencing results in increased sensitivity to tamoxifen in vivo and in vitro. Tamoxifen sensitivity of mouse tumor cells expressing Fliz1 will be compared to those in which Fliz1 expression has been abrogated. The cells will be inoculated into the mammary fat pads of syngeneic BALB/c mice, and sensitivity of tumor growth to tamoxifen will be assessed. IMPACT: The proposed studies will determine if Fliz1 regulates GATA-3 expression in mouse mammary cells. If Fliz1 expression contributes to the loss of GATA-3 expression seen in invasive human breast cancer, a potent therapeutic target is presented which could increase tumor sensitivity to hormone therapy and decrease metastatic potential.
The proposed study will explore the possibility that important clinical indicators of breast cancer prognosis and sensitivity to endocrine therapy are regulated by a novel protein factor, Fliz1. Fliz1 has not been studied in breast cells or tumors, but is hypothesized to inhibit production of a key protein, GATA-3, which is involved in making the estrogen receptor and in limiting tumor invasiveness. The studies may help define a new therapeutic target in breast cancer.