Six1-Eya-Dach Network in Breast Cancer

Principal Investigator: FARABAUGH, SUSAN
Program: BCRP
Proposal Number: BC073439
Award Number: W81XWH-08-1-0332
Funding Mechanism: Predoctoral Traineeship Award
Partnering Awards:
Award Amount: $96,947.14


I am very interested in how deregulation of gene activity in the cell induces abnormal growth in the breast and in ways to target novel cellular mechanisms for drug design in order to increase breast cancer treatment options. In my training, I will be working alongside a graduate student who is studying the 3-dimensional structure of the interaction between Eya proteins and Six1, a protein overexpressed in 50% of primary breast cancers and 90% of metastatic lesions. Our combined investigation of how Eya and Six1 are interacting, through structural and biochemical analysis, will lead to a greater understanding of how to block tumor promotion by the Six1 protein. As a breast cancer graduate student, my goal is to learn to think critically while gaining a strong foundation on which I can build in further continuing a breast cancer research-based career in either academia or industry. During normal development, genes are activated to stimulate cell proliferation, cell migration and invasion, and to regulate cell survival. These genes are usually downregulated once organ development is complete. In cancer, the same genes are often re-activated, stimulating the aforementioned properties out of context. The homeobox gene Six1 is such a gene. The Six1 transcription factor plays a critical role in normal development of the eye, inner ear, kidney, thymus, and skeletal muscle. Six1 expression is low or undetectable in normal adult breast tissue but is overexpressed in 50% of primary breast tumors and 90% of metastatic lesions. Overexpression of Six1 in the mammary gland has been linked to enhanced cell growth, tumor initiation, increased tumor volume, and metastasis. Additionally, overexpression of Six1 in normal mammary epithelial cells (MCF12As) induces numerous cancer-associated properties including proliferation, migration and invasion, and tumor formation. The ability of Six1 to activate gene expression depends on the presence of co-factors. These proteins regulate the function of Six1, defining whether Six1 is acting to repress or activate gene expression. The Eya family (Eya1-4) has been identified as co-activators of Six1, and the interaction between Eya and Six1 is known to be important for regulating genes that control cell growth during development. As Six1 is overexpressed in breast cancer, it is likely that Eya co-factors are required to complex with Six1 and activate cell growth and other developmental properties of Six1 out of context, leading to breast cancer. In this proposal, we will determine if Eya family members (Eya1-4) are required for the tumor-promoting function of Six1 in the mammary gland. We have recently demonstrated that Eya2 is the only Eya present in the MCF12A normal mammary epithelial cell line, and thus the MCF12As will be an ideal model to study the effect of Eya on Six1-induced tumorigenic properties.

The first aim of our grant will examine whether Eya2 is interacting with Six1 in mammary epithelial cells, as previously shown in other systems. In the second aim, we will examine whether Eya2 is necessary for driving the Six1-induced tumorigenesis seen in the MCF12As by removing Eya2. In the third aim, we will investigate the relevance of Eya2 as the Six1 co-activator in breast tumorigenesis by evaluating expression and function in a range of human breast tumor samples. To verify the importance of Eya2 in human breast cancer, we will correlate the expression of Eya2 with expression of Six1 in human patient samples and evaluate how combined expression affects clinical parameters such as stage, grade, and patient survival. Through this research, we hope to identify whether Eya2 is a necessary co-regulator of Six1 for the promotion and progression of breast cancer. It has been shown that removal of Six1 decreases tumorigenic properties such as proliferation AND metastasis. However, as a DNA binding protein, Six1 itself will not be easy to target with small molecule inhibitors. In contrast, the Eya proteins contain drug-targetable enzymatic domains that are required for Eya to activate Six1 and, as such, are very realistic targets for small molecule inhibitors. As Six1 and Eya proteins are developmentally restricted and are thus not present in most normal adult tissues, designing treatments to target the activity of the transcriptional complex would be expected to inhibit breast cancer on multiple fronts while leading to limited side effects. Therefore, understanding the role of Eya proteins in breast cancer has the potential to lead to novel therapies that could impact most breast cancer patients, since Six1 is overexpressed in a large percentage of breast cancers.