Analysis of a Protease Involved in Mammary Development

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Rana Zahedi, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7FB-0152 Award: $79,595
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2001)
Normal cells including breast cells, receive instructions from their environment telling them to divide, to create a tissue, or to perform specialized functions. Cancer arises when cells break away from the normal constraints on their behavior, and begin to grow in an uncontrolled and disorganized manner. To understand why cancer cells behave differently, it is necessary to understand how normal cells respond to their microenvironmental cues. Important information comes from other cells surrounding the normal epithelial cells. One of them is a signaling molecule called epimorphin. Epimorphin induces epithelial cell rearrangement. In breast , depending on how this molecule is presented to the epithelial cells, it induces different tissue arrangements. When presented to the epithelial cells (cells that can specialize to produce milk) only on one side it causes formation of long tubules that branch. When it is presented all around these cells, it triggers a globular like structure similar to those that make milk.

A thorough understanding of normal tissue architecture and development of breast is a crucial step before design of efficient and specific treatments for breast cancer. In order for different tissues and organs to be formed correctly, the cells need to receive signals which tells them what to do: whether to divide, migrate or stay put. Epimorphin is one of such signal molecules telling cells how to organize to assume the shape of the breast. There are other proteins (enzymes) that are called "proteases". These can cut proteins and provide additional signals. We have reasons to believe that during pregnancy a protease gets activated and cuts epimorphin. The cleaved epimorphin then give different signals to cells make them reorganize differently and form milk producing structures. To characterize the protease, several specific blocking reagents called protease inhibitors will be used. A biochemical approach will be also used to isolate the active protease based on its physical characteristics such as size, charge and solubility. If the protease is not known, its gene will be cloned and antibodies will be made for further characterization. The effects of high levels or absence of the protease on organization of our cell culture model that imitate the function of the mammary gland, and if times permit, in mice will be analyzed.

The mechanisms involved in the formation of alveoli (milk producing unit of breast) are not understood. This work will result in an understanding of how the information for formation of the breast unit is transmitted and will shed light on some aspects of this specialization and reorganization in normal breast, and how it may go wrong in cancer.

Final Report (2004)
We previously observed that epimorphin, a membrane bound morphogenic protein, was involved in various aspects of mammary gland development including branching versus alveolar structure formation. Our results suggested that to induce alveolar formation, epimorphin has to get cleaved and released from the cell surface by a metalloprotease. We proposed to characterize and identify the protease(s) involved.

Early in the project I identified MT-1 MMP as the potential protease-cleaving epimorphin. Therefore, I focused on determining when this protease was present in the mouse at different stages of mammary gland development including 7-week nulliparous, gestating, partuition, lactating and involuting glands. RTPCR on RNA isolated from mouse mammary showed a continuous expression of this protease. However we found that its expression appeared to be the highest in the 7-week-old nulliparous mice and during lactation. In collaboration with Dr. Mori, a post-doc at Dr. Bissell’s lab and an expert in membrane bound metalloproteases, we further analyzed the over expression of this protease in different phenotypically normal cell lines we had available in our laboratory.

While performing these studies I became interested in EMMPPRIN (Extracellular Matrix MetalloProtease Inducer). EMMPRIN is a membrane bound glycoprotein, which has been shown to induce the synthesis of several MMPs including MMP-2, MMP-3 and MT-1 MMP. EMMPRIN is highly expressed in tumor cells and is involved in tumor progression by inducing MMPs. I was interested in whether EMMPRIN was involved in normal mammary development. Therefore, I looked at the expression of EMMPRIN and these MMPs at RNA and protein levels. RTPCR on RNA isolated from mice at ages of 3, 5, 7, 9 and 11 weeks of age shows a continuous expression of EMMPRIN and MMP-2. MMP-3, however, appears to be regulated as it starts to be expressed at week 3, increasing at week 5, the strongest expression being observed at week 7 (the time when lateral branching occurs), and decreases afterwards back to week 3 levels. Immunohistochemistry analysis of EMMPRIN confirms that the protein is present and is expressed by the luminal epithelial cells. MMPs’ expression and localization are under investigation; technical difficulties have not allowed me to confirm their localization yet.