Heide L. Ford, PhD
2012-2013 BCRF Project:
(American Association for Cancer Research)
Division of Basic Reproductive Sciences
Department of Obstetrics and Gynecology
University of Colorado
In 2012-2013, Dr. Ford will continue research on the Six1/Eya complex, which plays a critical role in the onset and progression of breast cancers but has never before been clinically targeted. Six1 expression is low or undetectable in normal breast tissue but it is over-expressed in 50% of primary breast tumors and 90% of metastatic lesions. Dr. Ford’s team has demonstrated in laboratory models that the over-expression of Six1 results in enhanced proliferation, transformation, increased tumor volume, and metastasis. Importantly, RNA interference against Six1 decreases cancer cell proliferation and metastases in breast cancer models. The Eya proteins are coactivators of Six1 that seem to “switch” the Six1 transcriptional complex from a repressor to an activator complex. The Six1-Eya interaction is essential for proliferation during embryonic development, and both Six1 and Eya2 have been independently implicated in the same types of cancer. Furthermore, coordinated over-expression of Six1 and Eya2 significantly correlates with a dramatically shortened time to relapse and metastasis, and with shortened survival in breast cancers. These findings suggest that Eya and Six1 cooperate to stimulate breast tumorigenesis and progression. Importantly, recent evidence demonstrates that Eya protein’s phosphatase activity is critical for breast cancer metastasis.
Based on these observations, Dr. Ford’s team formed the hypothesis that the Six1/Eya complex is an ideal drug target whose inactivation will inhibit breast tumor cell proliferation and metastasis. They have collected data since the beginning of their research assessing the ability of the Six1/Eya complex to inhibit tumor cell proliferation and metastasis in breast cancers. Dr. Ford will continue testing Six1/Eya inhibitors for their ability to stymie tumor growth and metastasis. Since Six1 and Eya influence multiple stages of the tumor formation process, targeting this transcriptional complex can potentially inhibit breast cancer both at early and later stages of disease progression. In addition, since Six1 and Eya2 are not expressed in most normal developed tissues, therapeutic agents targeting the Six1-Eya complex should cause limited side effects and may provide significant benefits to the 50-90% of breast cancer patients whose tumors over-express Six1.
Mid-year Progress: Cancer and normal development share many properties. During normal development, genes are activated to stimulate cell growth, migration, invasion, vascularization, and to alter cell survival. These gene products are often lost once organ development is complete. In cancer, many developmental genes are re-activated, stimulating the aforementioned processes out of context. The Six1 gene encodes a transcription factor that induces the expression of a large number of genes that are involved in the growth, survival, migration, and invasion of cells during embryonic development. In most tissues, Six1 expression is lost once development is complete. However, Six1 is re-activated in many breast cancers, where as many as 50% of primary tumors and 90% of metastatic lesions express increased levels of the gene. During the two years of this award, Dr. Ford's team carried out experiments to test the hypothesis that because Six1 and its Eya cofactors are expressed during organ development, lost in most adult tissues, and re-expressed in tumors, the Six1/Eya transcriptional complex is an ideal drug target whose inactivation will inhibit tumor cell growth and metastasis with limited side effects. Towards the goal of testing this hypothesis, the researchers have identified a class of small molecules that target an enzymatic activity of Eya (the phosphatase activity, which removes phosphates from proteins). They continue to test the compounds in cell-based assays, including assays to measure the effects of the compounds on Eya mediated motility and cell growth. Furthermore, they have analyzed the mechanism of action by which the Eya phosphatase inhibitors work and shown that they directly bind to Eya. In addition, Dr. Ford's team solved the molecular structure of Six1 bound to Eya and demonstrated experimentally that the interaction between the two proteins is required for the ability of Six1 to promote tumor initiation and metastasis. The investigators then performed both rational based drug design and novel high throughput screens, and identified inhibitors of the interaction between Six1 and Eya. Some of these inhibitors have been shown to be efficacious in secondary protein-protein interaction assays, and Dr. Ford and colleagues are now characterizing these inhibitors for mechanism of action and efficacy against Six1-mediated pro-tumorigenic and metastatic phenotypes in cell culture.
Dr. Ford has been working on the role of the Six1 homeobox gene in tumorigenesis and metastasis since the late 1990s and has spent many years elucidating the mechanisms by which Six1 contributes to tumorigenicity and metastasis. Dr. Ford's main focus has been on understanding the numerous molecular mechanisms by which Six1 alters cell proliferation, cell survival, the epithelial to mesenchymal transition, and cancer stem cell characteristics, leading to its ability to induce both tumorigenesis and metastasis.
Dr. Ford's laboratory was the first to observe an overexpression of the developmental regulator Six1 in any cancer, and since then her group as well as many others have implicated it in both initiation and progression of many different tumor types. In addition, in the past few years Dr. Ford's team has dedicated much effort to understanding the role of the Six1 cofactor Eya, in mediating Six1-induced tumorigenic and metastatic phenotypes.
Dr. Ford's laboratory has published numerous papers (in journals such as Journal of Clinical Investigations, Proceedings of the National Academy of Science, Cancer Research, and others) that revolve around the role of the Six1 homeobox gene in tumorigenicity and metastasis. Many of their publications include work in transgenic, knockout, or xenograft models. In addition, Dr. Ford's recent focus has been almost entirely on the metastatic process, which is a significant theme in the project that is funded by BCRF in 2011-2012, as this research team would like to develop drugs that inhibit this process.
Based on the above observations, Dr. Ford's team therefore has formed the hypothesis that the Six1/Eya complex is an ideal drug target whose inactivation will inhibit breast tumor cell proliferation and metastasis. Because Six1 and Eya2 are embryonic genes with very limited expression in the adult, inhibitors of their expression/activity are likely to have limited side effects. In 2011-2012, the researchers aim to identify small molecules that inhibit the Eya phosphatase activity and small molecules that inhibit the Six1/Eya interaction. The accomplishment of these goals will further enhance the establishment of Six1/Eya complex as a new drug target.