Abstract. Work from the past 30 years has established the importance of epigenetic mechanisms of silencing of tumor suppressor genes. DNA methylation has emerged as one of the most common and conserved methods of epigenetic silencing, causing loss of expression of thousands of genes. Epigenetic alterations are significant drivers of tumorigenesis in all cancer types. Work from our group and others over the last 25 years has firmly established that specific genes that become aberrantly methylated uniquely, early, and frequently in breast cancer provide excellent biomarkers for breast cancer detection, prediction of response to therapy, and prognostication of the course of the disease. In the parent grant, we have proposed a five-gene panel for accurate detection of breast cancer in fine needle aspirates of suspicious breast lesions. Among them, Zinc finger (ZF) 671 (ZNF671) is a powerful detection marker, since it is frequently methylated in breast cancer and a variety of other carcinomas, but not in normal tissues. However, literature on its function is sparse; little is known about its upstream regulators or its downstream targets. For this supplement, we will investigate the pathways through which ZNF671 functions. We hypothesize that ZNF671 will behave as a tumor suppressor protein in breast cancer cells, both in cultured cells and in vivo. Based on the function of other ZNF proteins as embryonic differentiation factors, we propose that loss of ZNF671 will lead to dedifferentiation, increased stemness, and acquisition of aggressive neoplastic properties by cancer cells. To test this hypothesis, we proposed two specific aims. In Aim 1, we will study the phenotypic and molecular changes brought about by overexpression and knockout (KO) of ZNF671 in 4 breast cancer cell line models. In Aim 2, since the ability for unlimited replication is dependent on the stem cell population, we will test whether the presence or absence of ZNF671 influences the population of stem cells in breast cancer cells both in vitro and in vivo. Through this proposal, the baccalaureate candidate will learn the scientific process, and that of interpreting and reporting results. She will learn a variety of techniques to analyze biological and molecular changes brought about by overexpressing or knocking down the expression of ZNF671 in breast cancer cells. Microarray analysis of breast tumor cells with KO of ZNF671, its data analysis, and validation of gene expression by RT-qPCR will provide a wealth of data on downstream effector pathways of ZNF671. To achieve career developmental goals of the candidate, a program of training and career development opportunities through coursework, talks, networking, peer mentoring, shadowing and conference attendance and presentation has been described. At the completion of this supplement, the candidate will be fully equipped to compete for entry into a MD, PhD program.