The Role of Gli3 in Mouse Embryonic Mammary Gland Formation

Institution: Childrens Hospital, Los Angeles
Investigator(s): Jacqueline Veltmaat, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10FB-0116 Award: $90,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2004)
Normal development of an organ, in this case the breast, can be considered to be a progressive specialization of groups of cells in exchange for loss of their capacity to divide. These processes are influenced by the communication between neighboring tissues. Breast cancer can be considered to be the reverse process. Moreover, it has become increasingly clear that tumors arise as a result of misregulation of proteins that play a role in normal development. Less is know about the mechanisms through which these proteins regulate normal or tumor development.

Mutant mice lacking the function of any of the proteins GLI3, FGF10 or FGFR2b have similar defects in breast development. Here we propose to study the role of Gli3 in cell specialization and cell division, as well as in tissue communication in the developing breast. In addition, we want to study whether Gli3 collaborates with Fgf10 and Fgfr2b in these processes. Understanding these mechanisms may lead to better understanding of breast cancer.

Gli3 has multiple functions, which are independent from each other, during the initiation of mammary gland development and in shaping the developing early gland. Gli3 interacts with Fgf10 and Fgfr2b during the initiation of mammary gland development in the mouse.

We will compare the number of dividing cells in developing normal and mutant breasts. We will look if there are differences in the size and shape of the developing breasts or if there is production of other specific proteins in the developing normal and mutant breasts. We will separate the tissues from normal or mutant breasts and exchange them. Subsequently, we will culture these 'reconstituted' breasts in the laboratory, to see if any of these will result in a normally developing breast. This will tell us in which of the tissues the function of Gli3 is essential for normal development, and whether or not the observed defect in shaping the breast is a consequence of a delay in the onset of breast formation.

We will identify how Gli3 is involved in maintaining the delicate balance between cell division and cell specialization in the mammary gland. Loss of this control is associated with the onset of cancer. No previous studies have reported a role for GLI3 in mammary gland biology and pathology, although its related proteins GLI and GLI2 have been associated with other types of cancer, and GLI2 has been associated with aberrant breast development. This underscores the need to determine if GLI3 has a role in mammary gland development and pathology, which is the focus of this study.

Final Report (2006)
In the past few decades, it has become clear that "tumor" genes are in fact "developmental" genes that are misregulated through mutation. With that as a rationale, and with identifying new breast cancer genes and their mechanism of action as a goal, I selected several mouse strains that have a defect in the onset of breast formation due to the absence of function of particular genes, such as Gli3 and Fgf10. Interestingly, while mice normally form 5 pairs of breasts, some but not all, of these pairs failed to form in these mutants. This observation is important, because it indicates that at different positions along the chest and belly, breasts use different genes for their development, and are therefore likely different in their susceptibility for breast tumors when a particular gene is misregulated. This difference should be considered when designing a breast cancer study for which often the fourth gland pair of the mouse is used as a model for the human breast.

Whereas the fourth breast pair does develop in mutants for Gli3 and Fgf10, the third pair does not. Interestingly, the third pair is in position very similar to the human breast. We found that in the absence of Gli3, the production level of Fgf10 remains too low to induce sufficient production of Wnt signaling in skin cells, which is associated with a change of their identity into breast cells.

Importantly, other researchers have previously associated too high levels of Fgf10 and Wnt signaling with breast cancer, which can be considered as a loss of breast cell identity. This strongly suggests that Fgf10 and Wnt signaling need to be kept at an optimal level to maintain a breast cell's identity. It may thus similarly suggest that too high levels of Gli3 may cause Gli3 to function as a breast cancer gene, which will be the focus of a future study. In addition, with this work, we have identified a possible explanation for Poland's syndrome, which is a pathology entailing underdevelopment of the breast and its supporting muscle on one side of the chest.

We also found that the second breast pair develops abnormally in Gli3 mutants. This starts with the delay in the acquisition of a breast cell identity by skin cells, followed by a delayed maturation of the surrounding skin stroma into breast-supporting stroma. This indicates that Gli3 mediates the communication between the skin and the stroma, to enhance their change in identity. We are currently determining how Gli3 mediates this communication, which is crucial to finding ways to balance the activity of Gli3 when it is misregulated in breast pathology.

Symposium Abstract (2005)
Jacqueline M. Veltmaat, Wendy van Veelen, Bradley Spencer-Dene, Lendy T. Le, Jean Paul Thiery, Saverio Bellusci.

The Saban Research Institute/Childrens Hospital Los Angeles/USC Los Angeles, CA; Curie Institute, Paris, France; Cancer Institute, London, UK.

The mouse mammary gland is often used as a model to study pathology of the human breast. Here, we focus on the role of the GLI3 molecule in the mammary gland of the mouse. In contrast to the single pair of mammary glands that humans develop, mice have five pairs of mammary glands. Pairs #1, 2 and 3 develop in the thoracic region, and pairs #4 and 5 develop in the groin. We have observed that mice lacking the function of the GLI3 molecule, do not form mammary gland pairs #3 and #5, and show delayed and impaired formation of mammary gland pair #2. Mammary gland pairs #1 and #4 seem unaffected. We conclude that GLI3 plays a role in local decisions of the skin to convert to mammary tissue. The differential requirement for GLI3 amongst the pairs of glands indicates that the different pairs of mammary glands may not have the same genetic 'barcode' for the onset of their formation. This may be relevant for the appearance of more than 1 pair of nipples or complete breasts in 1-5% of the human population, most often near the normal breast, but occasionally in the groin. Importantly, these additional breasts are susceptible to the same pathology as normal breasts, including breast cancer.

Similar, but not identical to the absence of GLI3, the absence of FGF10 leads to the lack of formation of mammary gland pairs #2, 3 and 5, and in addition, the lack of formation of pair #1. We have found that the effect of the absence of GLI3 on mammary gland pair #3 is mediated via a reduction of FGF10 expression, whereas such a mechanism appears less or not relevant for the effect on mammary gland pairs #2 and #5. Our data show furthermore that the impaired formation of gland pair #2 starts with a delay of the conversion of skin tissue into mammary epithelium (eventually the milk producing tissue). In turn, the maturation of the surrounding stroma (predominantly fat tissue) is delayed and this is accompanied by an aberrant shape of the breast. This indicates that GLI3 is involved in the communication between the epithelial and stromal compartment of the breast, which is required for the normal development of the breast.

Our data indicate that the pairs of mammary glands have a differential requirement for GLI3, and that GLI3 plays multiple roles in the normal development of the breasts. Deregulation of genes that regulate normal development of a particular organ is often a cause of cancer. Therefore, our data suggest that each pair of mammary glands may also have a different genetic 'barcode' for the development of breast cancer. This difference may be relevant for studies using the mouse breast as a model for human breast cancer.