Cell Growth Control of Breast Epithelial Cells

Institution: Scripps Research Institute
Investigator(s): Ulla Knaus, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6JB-0063 Award: $265,950
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
The normal breast consists of fatty tissue with embedded ducts. The ducts of the breast are lined with milk-producing cells called epithelial cells, which are the origin of 80% of all breast tumors. The growth of breast epithelial cells is usually controlled by a balance between growing and dying cells. If this balance is disturbed and growth processes are favored, the first step towards breast cancer development has occurred. Breast cancer cells usually acquire several changes in their genetic material during cancer progression, which makes it difficult to evaluate the importance of one of these changes for cell growth. To determine if one specific, deregulated factor is a key element in cell growth control, its function in normal breast epithelial cells has to be examined.

We have recently discovered that a protein, which is implicated in aspects of cellular growth, is consistently active in human breast cancer cells and tissues. In these cancer cells this protein, termed Rac3, is required for cell growth. The same cancer cells possess a highly similar protein, termed Rac1, which is not active and localized in a different part of the cell. In this project we will investigate if growth control in breast epithelial cells is a specialized feature of Rac3, independently of Rac1. Normal breast epithelial cells grow in response to changing levels of growth factors and hormones. The influence of these stimuli on the activation of Rac3 and Rac1 in normal breast cells will be determined with a novel assay, which detects only the active form of the proteins and allows a distinction between these proteins. Activation of Rac3, but not Rac1, by certain stimuli will link Rac3 to specific cellular functions. We will define the role of Rac3 in these functions more precisely by determining in which part of the cell active Rac3 is present. Initial experiments with broken cells will be complemented with studies in living breast cells.

Our project has important implications for the development of treatments for breast cancer. Fast growing breast cancer cells have a need for deregulated, persistently active Rac3, but not Rac1. If Rac3 were a specialized, essential control protein for cell growth of normal breast epithelial cells, this protein would be a promising target for inhibitor development. Inhibitors directed against the differences between Rac3 and Rac1, either in their structure or in their localization in the cell, might decrease cell growth without affecting other functions.

Final Report (2003)
The development of both the normal breast and breast cancers is regulated by several stimuli including hormones and growth factors. Understanding the basic biology of normal breast cells will aid in the discovery of molecular mechanisms leading to pathologic conditions.

The regulation of cellular growth is under control of small GTPases, mainly of the Ras and Rho family. Two proteins, termed Racl and Rac3 are implicated in growth-regulatory processes. While these proteins are highly similar in their structure, their functions seem to differ substantially. Our goal was to define the localization of these proteins using cell separation methods and fluorescence microscopy. The localization in different compartments of the cell permits differential activation of the GTPase and interaction with a specific subset of other cellular proteins, and therefore determines the biological function.

To investigate the localization of Racl and Rac3 we introduced fluorescent copies of these proteins into epithelial cells. This allows us to detect the proteins either on special filters after cell fractionations or on slides by fluorescence microscopy. We did not see pronounced differences in localization of Racl versus Rac3 with the first method, but observed the distinct attachment of Rac3 to membranes inside the cell when using microscopy. In contrast, fluorescence was evenly distributed using Racl, indicating that Racl is a soluble protein in these conditions. Furthermore, we observed that several growth stimuli, which are known to activate the GTPase Racl, were ineffective on Rac3. We determined that Rac3 binds to a particular cellular structure termed Golgi and identified a small region in the Rac3 protein, which conveys this specificity. This information provides clues about the biological function of Rac3 and we have begun to analyze proteins, which associate with Rac3 at these internal structures and may play a role in growth control. A better understanding of the role of growth-regulatory GTPases in normal breast epithelial functions will have important implications for the development of treatments for breast cancer.