The primary cause of mortality in breast cancer is metastasis. Our research plan is focused primarily on the transcriptional lineage regulator factor, GATA3. The GATA3 gene is the third most mutated gene in breast cancer. GATA3 serves as a master regulator of breast epithelial luminal differentiation and maintenance and the GATA3 regulatory networks are deregulated during cancer progression. GATA3 status is an important prognostic factor in breast cancer. The loss of GATA3 plays an essential role in the loss of differentiation and the malignant conversion of breast cancer and the acquisition of metastatic capability. Well-differentiated tumors with high GATA3 expression have a lower propensity for metastasis, whereas poorly differentiated tumors with low GATA3 expression have a greater propensity for metastasis. Low GATA3 expression is strongly associated with higher histologic grade, positive lymph nodes, larger size, ER and PR negative status, and HER2 overexpression and a poorer prognosis. We have demonstrated that GATA3 is a suppressor of metastasis though its regulation of the differentiation state of the tumor cells as well as that of the tumor microenvironment. The goal of our research project is to define the molecular mechanisms through which the transcription factor GATA3 exerts its effects in regulating breast cancer metastasis. In Specific Aim 1, we determine mechanisms regulating GATA3 expression. In our preliminary studies, we have identified Zeppo2 (Zpo2, ZNF503) and ZBTB32 as regulators of GATA3 expression. We will focus on their roles as inducers of mammary tumor differentiation, progression and metastasis. Although most breast tumors express GATA3, tumor cells with decreased GATA3 expression have a more undifferentiated, basal phenotype and a higher metastatic rate. In Specific Aim 1, we will use chromatin immunoprecipitation followed by sequencing (ChIP-Seq) analysis to discover GATA3 targets in both mouse and human breast cancer. We will then investigate the mechanisms by which these GATA3-target genes contribute to GATA3's regulatory functions. In Specific Aim 2, we will investigate how the transcriptional network regulating GATA3 expression, focusing on the ZNF503/Zeppo2 axis. Since GATA3 is a critical regulator of breast cancer differentiation and suppressor of metastasis, then understanding these functions in molecular detail could lead to biomarkers and new therapeutic approaches.