In my predoctoral training, I will focus on the intersection between development, miRNAs, and breast cancer metastasis. miRNAs are naturally occurring small non-coding RNA that regulate gene expression in numerous contexts, including normal development, and are emerging as major contributors to cancer etiology due to their involvement in important processes such as cell growth, cell survival, migration, and invasion. Because breast cancer patients die from metastatic disease and not from their primary tumors, as a graduate student I will work intently to uncover possible mechanisms by which misexpression of an important developmental regulator, Six1, can alter miRNA expression and lead to breast cancer metastasis. In this process, I will gain considerable knowledge and the necessary expertise to become an independent breast cancer researcher in an academic setting. I am truly excited to have this unique advantage to study such an important area of breast cancer research, and I hope that I can build upon these experiences to ultimately contribute not only to the understanding of breast cancer, but eventually to the development of novel therapies to treat this deadly disease.

As eluded to above, my research focuses on the role of the developmental regulator, Six1, in breast cancer metastasis. Six1 is expressed at high levels during the development of the embryo where it mediates cell proliferation, migration, and survival. After development, Six1 levels significantly diminish in most adult tissues; however, when Six1 is re-expressed in the adult, it can mediate the aforementioned properties out of context, contributing to the onset and progression of cancer. Strikingly, Six1 is overexpressed in as many as 50% of primary breast cancers, but an even greater 90% of metastatic lesions. Importantly, Six1 overexpression in breast cancer also correlates with shortened time to relapse, shortened time to metastasis, and decreased survival. These data suggest that Six1 is an important regulator of metastatic disease. Indeed, overexpression of Six1 in a mouse model of breast cancer induces metastasis, providing conclusive evidence that Six1 is a metastatic regulator. We have further shown that Six1 may induce metastasis via its ability to induce an epithelial-to-mesenchymal transition (EMT), a process that is often correlated with increased metastases. The induction of EMT by Six1 is dependent on its ability to induce TGF-beta signaling, a critical pathway in both EMT and metastasis. TGF-beta is a multifunctional growth factor, which serves a dual role in tumorigenesis. In early-stage cancers, TGF-beta signaling is believed to be tumor suppressive, via its ability to cause growth inhibition. However, in later stage tumors, the growth suppressive functions of TGF-beta are lost, and it instead promotes tumorigenesis through activating an EMT. Interestingly, Six1 overexpression enhances the EMT promoting function of TGF-beta, while diminishing the ability of TGF-beta to cause growth inhibition.

My project will test the hypothesis that Six1 stimulates breast cancer metastasis via its ability to activate the tumor promoting functions of TGF-beta while inhibiting the tumor suppressive functions of TGF-beta. We will examine whether Six1 influences TGF-beta signaling via its ability to regulate miRNAs 106b, miR-93, and miR-25, three miRNAs that are clustered together in the genome. The miR-106b-25 cluster was identified in a miRNA microarray screen in which we were looking for miRNAs regulated by Six1. Further validation studies demonstrated that Six1 regulates the expression of these cluster miRNAs in mammary carcinoma cells. Interestingly, recent literature demonstrates that these miRNAs may be involved in overcoming growth inhibition mediated by TGF-beta, and our bioinformatic analyses suggest that they may also induce the EMT-promoting aspects of TGF-beta signaling. Thus, these miRNAs may provide an explanation for how Six1 overexpression leads to the activation of TGF-beta signaling that results in EMT, and ultimately metastases. In this project, it is my goal to examine these cluster miRNAs as a mechanism by which Six1 mediates its protumorigenic and metastatic functions in breast cancer, with an emphasis on their regulation of TGF-beta.

The impact of connecting miRNAs to Six1-mediated breast cancer metastasis has enormous implications in the further treatment of breast cancer. miRNAs hold remarkable promise as diagnostic tools due to their stability in vivo as compared to mRNAs, and detection of these molecules in patients can be used as prognostic markers for breast cancer. Additionally, miRNAs, if identified as tumor promotional, become good targets for novel anti-cancer agents, as the inhibition of one miRNA may have an impact on multiple processes by affecting the expression of numerous gene products. miRNAs induced by Six1 may make exceptionally good anti-breast cancer targets, particularly if, like Six1, they are not expressed in most adult tissues but re-expressed in tumors. In this case, targeting these miRNAs for anti-breast cancer therapy has the potential to inhibit metastasis while having limited side effects. Indeed, such breast cancer-specific therapies are badly needed.