A Genetic System for Identification of Mammary Stem Cells

Institution: Salk Institute for Biological Studies
Investigator(s): Dannielle Engle, BA -
Award Cycle: 2009 (Cycle 15) Grant #: 15GB-0015 Award: $76,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2009)

It has been proposed that mammary stem cells or cells with similar properties to mammary stem cells contribute to breast cancer. These so called mammary “cancer stem cells” have been speculated to be quiescent or possess drug efflux pumps, which would make them resist to commonly used chemotherapeutic agents. Thus, current treatments might temporarily reduce tumor bulk, but leave dormant cancer stem cells to regenerate the tumor upon their reentry into the cell cycle. If this hypothesis is correct, identifying cancer stem cells may allow earlier detection, more accurate prognoses, and development of potentially curative therapies. However, current technology and strategies yield mammary stem cells that are not sufficiently pure to enable their characterization. To overcome this obstacle, we developed a novel genetic approach to enable rapid characterization of mammary stem cells using signaling pathways these cells likely require. Successful completion of the project would enable functional identification of stem cells in the normal mouse mammary gland and mouse models of breast cancer.

My first aim is to gain a better understanding of the developmental pathways involved in mammary development, and to localize the cells in which these pathways are active. It was previously demonstrated that Wnt signaling is critical for mammary development. Wnt proteins comprise a family of highly conserved secreted molecules that regulate proliferation, maintenance of stem cells, and many other functions. Therefore, I will characterize the expression of Wnt signaling pathway components during mammary development by analyzing the expression of several Wnt target genes. I will also study p63 (a member of the p53 tumor suppressor family), integrins (cell adhesion proteins), and other proteins thought to be important for mammary formation. I will perform these analyses using immunochemical techniques. To test whether Wnt signaling is involved in mammary stem cell function, we developed a genetic strategy using live cell fluorescence with a Wnt-inducible gene promoter to track, purify, and characterize individual cells in mouse embryos and adults. This system enables us to determine whether the cells with active Wnt signaling are dividing or are quiescent. Importantly, if the stem cells are not in cycle, our genetic strategy will enable us to identify and isolate them and to then test whether particular growth pathways affect their functionality using transplantation assays.

Existing studies of mammary and other tissue stem cells have used isolation techniques that rely on the expression of proteins (CD markers) on the surface of the cells. Using this technique, it has taken 20 years to purify blood stem cells to a level of only about 50%, while that of mammary stem cells is only 2-5%. Such a low level of purity makes it difficult to pinpoint the identity of mammary stem cells, as their unique features are masked by the contaminating cells. We hope that our new mouse model-based technology will ultimately be translated into useful human, clinical applications by definitively determining whether mammary stem cells or cells with similar properties contribute to breast tumorigenesis.

Final Report (2011)

Mammary stem cells (MaSCs) have unique properties during development that may be relevant to the detection and treatment of breast cancer. Isolation of these cells from embryonic day 18.5 (E18.5) resulted in a higher level of purity than achievable using the same strategy in the adult gland. Studies of MaSCs during fetal development revealed many candidate pathways with functional relevance for this population in vitro that may be applicable to tumorigenesis. For example, unlike their adult counterparts, spheres generated from this population enriched for fMaSCs express activated ErbB family members and are sensitive to ErbB signaling inhibition.

We evaluated the level of heterogeneity within the fMaSC- enriched population using single cell qRTPCR. These analyses revealed that there exist two cell types within the fMaSC-enriched population. The presence of a neuronal like cell type was found to be dispensable for fMaSC function at E18.5, but required for adult MaSC function by transplantaiton analyses. Interestingly, there may be a functional role for this cell type in inducing MaSC activity in homeostatic states and before MaSC specification has occurred.

Finally, we generated a universal acceptor site within the Rosa26 locus of mice to facilitate the rapid generation of genetic reporters with the ultimate purpose of tracking MaSCs during development and disease. The homologous recombination was successful and we successful established mouse colonies with the universal acceptor locus. Upon isolation of pristine ES cells from this line, we will introduce a Wnt dependent, label retention system into this locus using CRE mediated recombination.

Symposium Abstract (2010)

Mammary development and tumorigenesis exhibit potentially important similarities. The growth of the mammary gland and its invasion through the stroma are reminiscent of the steps involved in tumor progression and metastasis. Mammary stem cells play a key role in the development and maintenance of the breast. These cells have the ability to self-renew and generate the different cell types of the breast such that an entire functional mouse mammary gland can be regenerated from a single mammary stem cell. Similarly, it has been shown that some tumors contain a population of cells with disproportionate ability to generate new tumors upon implantation in mice. As these cells can replicate and generate the same cell types present in the primary tumor, they have been operationally termed cancer stem cells. In addition to these two stem like characteristics, cancer stem cells are also thought to be resistant to cancer therapies. Therefore, if even a single cancer stem cell survives after the treatment of a tumor, it could result in cancer relapse. Unfortunately, pure populations of mammary stem cells or breast cancer stem cells have not yet been identified, making it difficult to determine which signals regulate stem cell activity, how cancer stem cells contribute to breast cancer, and whether cancer stem cells can be effectively targeted and eliminated. Using properties important for development of the mouse mammary gland and stem cell function, my research focuses on improving mammary stem cell identification, isolation, and characterization.

It has been proposed that some tissue stem cells exist in a dormant state. I will introduce an artificial gene into mice that permits visualization of cells with different rates of division. In addition, mammary gland development has been shown to rely on Wnt signaling, a pathway that is also critical for stem cell function in several other tissues. For this reason, the artificial gene is designed to also label cells responding to Wnt. Before generating these mice, I confirmed the artificial gene is functional by stimulating Wnt signaling in vitro. Once I have finished generating these mice, I will be able to test my hypothesis that mammary stem cells exhibit and rely on these two features to maintain their activity by assessing the ability of Wnt responding cells with different rates of division to re-grow a mammary gland after transplantation. In addition, I setup a culturing system that recapitulates mammary development in order to establish a parallel in vitro model for use with this mouse. Using this in vitro model, I am optimizing modulation of Wnt signaling to test its effect on mammary stem cell activity. Ultimately, utilizing the genetic system during development to isolate cells based on their functional properties, we will be able to isolate mammary stem cells and determine what signals are essential for stem cell activity during development and tumorigenesis. Understanding the molecular signals that govern normal mammary development and mammary stem cell activity is likely to provide additional markers for early detection, targets for treatment, and inroads to the prevention of breast cancer.

A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer.
Periodical:Cell Stem Cell
Index Medicus: C Stem Cell
Authors: Spike BT, Engle DD, Lin JC, Cheung SK, La J, Wahl GM
Yr: 2012 Vol: 10 Nbr: 2 Abs: Pg:183-97