One in nine women will develop breast cancer sometime in her life, and 90%-95% of these breast cancers will arise sporadically. Various risks associated with the disease include early onset of menarche, late age of menopause, nulliparity, or having a first full-term pregnancy after the age of 30. Mammary gland development primarily occurs post-embryonically, undergoing cellular growth, expansion, extensive remodeling, and differentiation in the adult mammal. Many of the changes that occur during normal development are often mimicked, inappropriately, during breast tumorigenesis. This suggests that breast cancer is likely influenced by genes important for developmental processes. Homeoproteins regulate the expression of numerous genes and act as "master" regulators of development, affecting cell proliferation and differentiation, cell death, neovascularization, cell motility, and invasion of surrounding tissue. The Six1 homeoprotein is dynamically expressed in the developing mammary gland, with the highest levels of expression in the embryonic mammary gland that decrease as the gland differentiates, until its expression is almost absent in late pregnancy and in lactation. This correlates with the expression pattern of Six1 observed in other organs during embryogenesis, where it is known to play a role in the proliferation of progenitor cells and is primarily lost in cells as they become more specialized. Interestingly, while Six1 expression is low in most differentiated tissues, its expression is increased in a number of cancers derived from tissues where it is believed to play a developmental role, including Wilms' tumors, rhabdomyosarcomas, and breast cancer. Six1 is overexpressed in up to 50% of primary breast cancers and 90% of metastatic lesions, and may contribute to tumor formation by re-initiating a program for proliferation that is normally only activated in the developing embryo. Studies have shown that Six1 overexpression is sufficient for converting normal mammary epithelial cells into tumor-forming cells in mice. Cyclin A1, a gene that is expressed during early embryogenesis, is controlled by Six1 and functions by stimulating the cell cycle, which leads to cellular proliferation. Our studies are going to focus on whether Six1 and cyclin A1 have a role in initiating breast cancer through re-activation an embryonic program for proliferation. Furthermore, because Six1 also has been implicated in metastasis (both in breast cancer and in rhabdomyosarcomas), our studies may extend beyond tumor initiation and also address issues of tumor progression. Because Six1 and cyclin A1 are not normally expressed in the adult animal, their function could be effectively targeted in breast cancer while avoiding many of the undesirable side effects normally associated with other chemotherapeutics.

In order to best simulate how normal breast cancer arises, we have engineered two animal-based models that will allow us to turn on and off Six1 expression in the mouse mammary gland at will. The first aim of our study will use one model to overexpress Six1 in all mammary epithelial cells when the animals are treated with doxycycline. The second model will be used to target progenitor mammary cells for Six1 overexpression. In both models, we will be able to see whether Six1 can cause tumorigenesis, alone or in combination with other oncogenes. We can then turn off Six1 expression after tumors develop to see whether they are dependent upon Six1 for maintenance. This will ultimately determine the relevancy of Six1 as a therapeutic target. Our second aim will use both models to determine whether Six1 is able to induce tumorgenesis when cyclin A1 is absent. These experiments will determine whether Six1 mediates tumorigenesis by re-activating an embryonic program of proliferation through cyclin A1 and will suggest whether Six1 and cyclin A1 are potential therapeutic targets for breast cancer.

Our models of Six1 overexpression should enable us to gain insight into how genes that are important in normal development can be "hijacked" to perform tumor-promoting functions later in life. Both transcription factors and cyclins have been successfully targeted to inhibit tumor growth, and as such, if we demonstrate the importance of Six1 and cyclin A1 in breast tumorigenesis, we can tailor our therapies on past successful therapies against similar types of molecules with the advantage that Six1 and cyclin A1 are not necessary for most normal adult cell survival, and thus therapies directed against them may not lead to the severe side effects seen with other, more conventional therapies. Together, our proposed animal model studies will begin to assess whether Six1, and its transcriptional target cyclin A1, may actually be such ideal therapeutic targets.