A New Marker for Mammary Epithelial Stem Cells?

Institution: The Burnham Institute for Medical Research
Investigator(s): Robert Oshima, Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12IB-0106 Award: $188,827
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2006)
Mammary epithelial stem cells capable of reconstituting a fully functional gland exist and are thought to be responsible for the remarkable recycling capability of the gland through multiple pregnancies. We have discovered a new marker gene that is expressed in several other types of somatic stem cells and wish to determine if it is also preferentially expressed in mammary epithelial stem cells. If so, we will use its expression to enrich or purify the normal mammary epithelial stem cell and compare its differentiation capability in vivo and in cell culture.

Our potential mammary stem cell marker gene is called maternal embryonic leucine-zipper kinase (MELK). MELK is preferentially expressed in several other stem cells (hematopoietic, neural, possibly in epidermal stem cells). These stem cells share a capability for self-renewal while retaining the potential for differentiating to different cell types. We will characterize expression of MELK during mammary gland development and test whether MELK expressing mammary cells have potential for reconstitution of a functional mammary gland. We will also compare mammary reconstitution capability in whole animals (mice) with mammary cell differentiation in three-dimensional cell culture. Our experiments will use excised mammary glands from the MELK-GFP transgenic mouse to examine the GFP and endogenous MELK RNA co-expression by histological methods. MELK-GFP epithelial cells will be isolated from virgin and pregnant animals using fluorescent activated cell sorting (FACS) and tested by transplantation into the cleared fat pads of 21-day old female mice of the same genetic background. We will also use cells in culture, grown in suspension to test the proliferative and differentiation potential of MELK-GFP cells.

Understanding how mammary epithelial stem cells become quiescent, but retain the potential for proliferation and differentiation over many cellular generations, is likely to be a key to understanding events that lead to breast cancer and cancer cell dormancy. MELK has already been identified as a potential target for intervention in many types of cancer. Understanding its specific role in stem cell self-renewal or differentiation may lead to strategies of inducing “benign differentiation” as an alternative to the current use of chemotherapeutics and other drugs, often in a futile attempt to eradicate tumor cells.

Final Report (2008)
During the year of support for this grant, we tested the behavior of mammary cells that were marked with a green fluorescent protein (GFP) both in mice mammary fat pad transplantation models) and in cell culture. Within animals engineered to express the GFP to mark where the maternal embryonic leucine zipper kinase gene was active, we found that the mammary cells that most express the protein were dividing cells that contribute to luminal, interior lining of the mammary ducts. We found that culturing the GFP expressing cells from single isolated cells resulted in spheroid colonies composed of at least two distinct cell types. However when the same cells were tested for the potential to generate a new mammary gland in animals, we found that they were not enriched for this stem cell activity.

A full description of Melk with a discussion of its potential role in various cell biological contexts is found on-line: http://www.signaling-gateway.org/molecule/query?afcsid=A003002

We have concluded that the cells expressing Melk are proliferative progenitors that have not preferentially retained the potential for reconstituting a new mammary epithelial tree. The identity of these cells with cancer stem cells remains to be determined. We are continuing these pilot studies and expect to publish the results in 2008.