Novel Breast Cancer Epithelial Cell Metalloproteinase

Institution: California Pacific Medical Center Research Institute
Investigator(s): Pierre-Yves Desprez, Ph.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3IB-0123 Award: $193,995
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (1997)
Breast cancer occurs as a result of alterations in the mechanisms that control normal cell behavior. Cancer and normal cells differ in two fundamental ways: (i) cancer cells grow inappropriately and (ii) cancer cells generally lose their ability to serve the normal functions. These two differences are closely associated with breast epithelial cells, the cell type from which more than 90% of the breast cancer arise. In addition, cancer cells may migrate (metastasize) from the breast and invade the lymph nodes and other foreign sites (e.g., lung or bone). Our research will provide critical insights into how breast epithelial cells lose their proper control of growth and function and, in particular, how they acquire the ability to become invasive to initiate metastasis.

We will use a breast cell culture system developed previously to carry out these studies. Using this cell system, we have already shown that a specific intracellular protein (Id-1) is critical for coordinating the growth and function of the cells. When the Id-1 level is elevated inappropriately, it causes the cells to lose their normal functions and become invasive. Further, we have found that cells with an elevated level of Id-1 protein will secrete an extracellular enzyme (a metalloproteinase), which can potentially destroy the extracellular matrix surrounding the breast epithelial cells. Therefore, this enzyme may be a crucial factor in making the breast epithelial cells abnormally invasive in cell culture, and it may also contribute to the invasive potential of some aggressive breast tumors. We will study this metalloproteinase enzyme by isolating it using two different approaches. After isolation, we will have both the protein sequence and the DNA probes, which will then allow us to answer several fundamental questions. For example, how is the novel metalloproteinase related to other described enzymes of different size and distribution? Where is this enzyme produced in the breast and other organs in the body? Is this enzyme overproduced in the breast of breast cancer patients? Our eventual objective is to determine whether this enzyme can trigger metastasis in breast cancer cells.


Final Report (2000)
Note: This grant was extended one year to complete the aims.

Breast cancer occurs as a result of alterations in the mechanisms that control normal cell behavior. Cancer and normal cells differ in several fundamental ways, (i) cancer cells grow inappropriately, (ii) cancer cells generally lose their ability to serve the normal functions, and (iii) cancer cells may migrate (metastasize) from the breast and invade the lymph nodes and other foreign sites (for example lung, brain and bone).

Using a breast cell culture system (called SCp2), we have already shown that a specific intracellular protein (Id-1) is critical for coordinating the growth and function of mammary epithelial cells. When the Id-1 level is elevated inappropriately, it causes the cells to lose their normal functions and become proliferative and invasive. Further, we have found that in cells with elevated levels of Id-1 protein, a secreted, extra-cellular matrix protease was produced. Our goal was to purify, clone, and study the properties of this novel, Id-1-regulated breast cell protein. We initially believed that this protein was a member of the matrix metalloproteinase (MMP) family, which are key regulators of breast cell invasiveness.

The principal strategy to clone this novel MMP was to use a conserved protein sequence present in many MMPs, and using the technique of Polymerase Chain Reaction (PCR)-amplification. These conserved MMP sequences allowed us to design degenerate PCR oligonucleotides that were expected to hybridize with most or all MMP cDNAs. Thus, we attempted to clone this unique MMP by PCR using these degenerate oligonucleotides as primers. One of the clones, a 1.6-kb PCR fragment, corresponded to an already cloned MMP, a specific MMP (membrane-type MMP) bound to the cell surface of invasive cells. We then used another set of primers, and this time we obtained a 2.6-kb fragment that did not show any homology with already known genes. Moreover, after sequencing from both ends, it contained a ‘zinc finger’ domain. This gene was differently expressed in control and Id-1transfected cells. We introduced this novel zinc finger protein into SCp2 mammary epithelial cells in order to determine its function. We found that it was able to trigger a higher growth rate. Therefore, even if this gene did not appear to be the MMP-like enzyme involved in invasion, but it may represent an important mediator of Id-1-induced proliferation in breast cells.

More research needs to be performed to determine the significance of this novel protein in breast cells and how it might be associated with breast cancer.

Molecular cloning and characterization of a zinc finger protein involved in Id-1-stimulated mammary epithelial cell growth.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Singh J, Itahana Y, Parrinello S, Murata K, Desprez PY.
Yr: 2001 Vol: 276 Nbr: 13 Abs: Pg:11852-11858