Role of Telomerase in Mammary Stem Cell Function

Institution: Stanford University
Investigator(s): Steven Artandi, M.D., Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0174 Award: $236,379
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2005)
Telomerase is expressed in unspecialized mammary stem cells and progenitor cells (i.e., cells that have begun the path to specialization as ductal, lobular, or myoepithelial cells), but its role in these compartments is not fully understood. The standard thinking on the function of telomerase in these stem/progenitor cells is to counter loss of DNA that occurs at the chromosomal ends with each cell replication cycle. Overexpression of TERT, the protein catalytic component of telomerase, in human mammary epithelial cells results in telomere elongation and cellular immortalization. Telomerase is known to be reactivated in 90% of human breast cancers, where it is thought to serve a similar function in endowing these tumors with unlimited proliferative potential. However, recent data from my laboratory challenges this paradigm and indicates that TERT possesses other activities that do not depend upon its well-understood function in elongating telomeres.

In this study we will test the hypothesis that conditional activation of TERT in adult mouse mammary epithelium leads to increased proliferation and breast cancer by way of promoting proliferation or expansion of mammary stem/progenitor cells. We will also use genetic means to determine if these effects of TERT involve telomere synthesis. The aims are: 1. To determine how conditional induction of TERT alters proliferation and development in the mammary gland. Using a novel tetracycline-inducible TERT system that we have engineered, we will activate TERT in the adult mammary gland to understand how TERT induces proliferation and breast cancer. 2. To understand how TERT modulates mammary stem cell function. We will test if activation of TERT leads to expansion of stem/progenitor cells and if TERT extends serial transplantation by enhancing mammary stem cell function.

We use a novel conditional transgenic system for inducing TERT in adult mammary epithelium with a tetracycline-regulated approach. Effects on mammary epithelium will be studied using whole mount (i.e., spreading and staining the whole tissue on glass slide) and histological (i.e., sectioning and microscopic) analyses. Proliferation will be assessed using BrdU incorporation and Ki-67 staining. Tumor formation will be studied by careful necropsy and histological analysis. The requirement for TERT's telomere synthesis function will be dissected genetically in a telomerase RNA component (TERC)-negative background. A potential effect on the mammary stem/progenitor compartments will be studied by Sca-1 and Keratin-6 staining, and by serially transplanting mammary epithelial into de-epithelialized hosts having their epithelial compartments removed.

A detailed understanding of TERT's multiple functions in mammary stem cell function and in breast cancer may lead to novel therapeutic approaches to block these functions. For example, current anti-telomerase strategies aim to inhibit enzymatic function. In contrast, a more effective strategy might entail inhibiting TERT protein, or other elements in a potential TERT signaling cascade.

Final Report (2007)
Telomerase is an enzyme expressed in stem cells and in cancer cells, but not in the vast majority of normal cells in human tissues. One function of telomerase is to add repeats to structures at chromosome ends, called telomeres. Telomeres are DNA repeats bound by a large complex of proteins. Telomeres protect chromosome ends from degradation and keep chromosomes stable. In breast cancer, there is growing evidence that telomeres shorten during early tumor development, but then stabilize when telomerase is upregulated (increased in abundance) at the ductal carcinoma in situ (DCIS) stage. We recently discovered that, in addition to its role in stabilizing chromosome ends, telomerase can activate quiescent tissue stem cells. Mammary gland, like most human tissues, is maintained by stem cells that can give rise to all epithelial cells within the organ.

To understand the role of TERT, the protein subunit of telomerase, in mammary tissue and mammary stem cells, in the first phase of this project we have successfully generated a genetically engineered mouse (GEM) system in which expression of TERT can be switched on and off using a drug (deoxycycline, a semi-synthetic tetracycline) that is administered in drinking water. By trying two independent mouse transgenic lines that we generated, we have found a combination that allows tight control of TERT expression in mammary tissue. Analysis of RNA derived from our mouse mammary glands shows that TERT is expressed in strictly a drug-dependent manner. After short periods of TERT expression, we do not detect an obvious effect on gland architecture. However, after sustained expression of TERT in adult mouse mammary gland, we see signs of cellular hyperproliferation (excessive growth) and early cancers. In subsequent studies, mice that we engineered to lack telomerase exhibit telomere shortening that impairs mammary gland development and side-branching during pregnancy. These findings have important relevance for understanding how the gland changes with aging and cancer development. Finally, to understand the role of TERT in mammary stem cell self-renewal, we have developed a transplantation assay where stem cells from one GEM mouse are transplanted into a normal recipient. We show that this procedure will reconstitute the mammary gland in the mouse that received the stem cells. When we knockout TERT, we initially observe that mammary stem cells function normally. However, if we breed TERT knockout mice through several generations to allow telomeres to become very short, we now find that the function of mammary stem cells is profoundly impaired.

These studies provide important insights for the role telomerase (TERT) plays in maintaining telomeres and preserving mammary stem cell function during normal development and maintenance of the organ, and likely, during tumorigenesis.