The incidence of breast cancer in women in the United States is 1 in 8 (13%), and it is the second-most common cause of cancer-related deaths in women (after lung cancer). There are many prognostic factors associated with breast cancer such as staging and tumor size, location, and grade. In addition to these factors, the presence of cancer cells in the lymph nodes is a strong marker of poor prognosis. Indeed, breast cancer patients will die from the spread of cancer to other parts of the body such as bone, brain, and lung via blood vessels or the lymphatic system, not from the growth of their primary tumors. I am particularly interested in studying breast cancer dissemination, because this leads to such significant morbidity and mortality for breast cancer patients. The focus of my studies is how misexpression of an important developmental regulator, Six1, leads to lymphatic metastasis in breast cancer. The training I receive will enable me to build up my knowledge in breast cancer biology, with a specific emphasis on lymphatic dissemination of tumor cells, and will allow me to begin to think about novel ways to treat and/or prevent breast cancer dissemination. I believe my training in the laboratory of Dr. Ford will contribute greatly to my long-term goal to become an independent breast cancer researcher.

The Six1 homeoprotein is a transcription factor that plays a critical role in normal development of the mammalian inner ear, kidney, thymus, and skeletal muscle. Its expression is high during embryogenesis, but it is largely undetectable in most normal adult tissue. This holds true in the mammary gland, where Six1 is highly expressed during embryogenesis, but is undetectable in normal, differentiated adult mammary tissue. Interestingly, Six1 is re-expressed in a high percentage of primary (50%) and metastatic breast lesions (90%). Importantly, its expression in breast cancer correlates with shortened time to relapse, shortened time to metastasis, and decreased survival. In addition, its expression in breast cancer samples highly correlates with lymph node positivity in three independent breast cancer studies (p=9.50E-04, 0.003, 0.01, respectively). To determine whether Six1 plays a causal role in metastasis, we developed a mouse mammary tumor model and demonstrated that Six1 overexpression does indeed lead to a 40%-60% increase in metastasis. The vast majority of Six1-induced metastases were found either in the lymphatics, or the lymph nodes, and interestingly, we found that Six1 induced the expression of a critical lymphangiogenic regulator, VEGF-C, in these tumors. Together, these data strongly suggest that Six1 overexpression in breast cancer promotes lymphatic dissemination and lymph node metastasis, perhaps through its ability to stimulate the formation of new lymphatic vessels.

In this proposal, we will determine whether Six1 induces lymphangiogenesis (the formation of new lymphatic vessels) and lymphatic metastasis, via its ability to upregulate a critical lymphangiogenic regulator, VEGF-C. VEGF-C is an important growth factor for the development of lymphatic vessels. Normally, lymphangiogenesis is quiescent under physiological conditions in the adult; however, growing evidence indicates that tumor cells can secrete factors such as VEGF-C to promote the growth of surrounding lymphatic vessels. This formation of new lymphatic vessels not only favors tumor growth (by offering nutrients and decreasing interstitial fluid) but also offers a way for the tumor cells to move away from the primary site, leading to metastases. Through this research, we will use in vitro (cell culture) and in vivo (animal) models to evaluate whether the Six1-mediated upregulation of VEGF-C is required for the formation of new lymphatic vessels (lymphangiogenesis) and for the ability of Six1 to induce lymphatic metastasis.

Understanding the molecular mechanism by which Six1 induces lymphatic metastasis may in the future allow us to develop either prognostic tests or therapeutic treatments that focus on Six1 or genes that it activates in an attempt to inhibit metastatic dissemination. Because Six1 is not expressed in most adult tissues, and because it plays a causal role in lymphatic metastasis (and in proliferation and survival of tumor cells), targeting it may be particularly beneficial as it should inhibit breast cancer metastasis, while sparing the normal cells. Such cancer therapies are badly needed, and it is for this reason that we are interested in understanding how Six1 induces breast cancer metastasis.