Regulation of Breast Epithelial Cell Motility by Proteases

Institution: Scripps Research Institute
Investigator(s): Vito Quaranta, M.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4PB-0162 Award: $464,030
Award Type: Request for Applications
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (1998)
Breast cancer is lethal because cancer cells are able to invade the surrounding tissues. Eventually, these cells grow at distant sites, and the spread of breast cancer throughout the body becomes life threatening. If one could prevent cancer cells from invading, breast cancer would remain confined and could conceivably be cured by surgical removal of the initial cancerous lesions. Our interest is focused on the very early step of cancer cell invasion, its crossing of the basement membrane. In the normal breast gland, the basement membrane keeps the breast epithelial cells neatly organized in functional structures. Importantly, it keeps them separated from the underlying connective or fat tissues. At early stages, even breast cancer cells are effectively segregated from surrounding tissues by the basement membrane. This is recognized clinically as carcinoma in situ. A critical transition in the development of breast cancer occurs when cancer cells no longer respect the boundary represented by the breast gland basement membrane. Once they invade the underlying tissues, cancer cells have made the first step to spread to the rest of the body. The transition to invading cells is unpredictable in all cancers, but particularly so in breast cancer. Therefore, there are several important questions to be addressed. What motivates cancer cells to cross the basement membrane? What are the mechanisms that make normal cells respect the basement membrane boundary? How do these mechanisms break down in invading breast cancer? An answer to these questions will form a foundation for novel treatment of breast cancer, specifically aimed at blocking cancer cell invasion and spread (metastasis).

With this proposal, we hope to identify one mechanism of breast cancer cell invasion, which may represent a target for drug discovery. We found that a substance called protease MMP2 acts upon a constituent of the basement membrane, called laminin-5 (Ln-5), and changes it into a stimulant for cell invasion. We showed that breast cells like to adhere to Ln-5 and stay put. In contrast, if breast cells come in contact with Ln-5 that has been modified by the protease MMP2, they become highly motile. It is perhaps more than a coincidence that, in tissue specimens from patients, MMP2 is often found concentrated along sites where breast cancer cell invasion occurs. We believe this phenomenon reflects a mechanism whereby MMP2 stimulates cancer cells to invade by activating a function on Ln-5 that makes cells move around. The basic knowledge about the breast obtained from these studies may form the basis for novel approaches for designing drugs that can stop breast cancer cells from spreading, even at advanced stages.

Final Report (2001)
The long-term goal of our research is to understand how breast cancer cells invade other tissues. One critical determinant of invasive cancer cells is the ability to travel through the body, generally referred to as cell migration. In this project, we tested specific hypotheses concerning precise molecular mechanisms that underlie cancer cell migration. We focused on the extracellular matrix (ECM), a network of macromolecules that surrounds cells and is itself the substrate through which cells migrate by responding to ill-defined chemical cues. A growing body of evidence suggests that matrix metalloproteinases (MMP) may generate some of these cues, by cleaving specific ECM components.

We surveyed invading cancer cell lines and found that inhibiting MMP activity consistently leads to reduction in migratory potential through laminin-5 (Ln-5), an important ECM component of the breast gland basement membrane. Profiling these cell lines for the presence of different types of MMPs revealed a strong correlation between spontaneous Ln-5 migration and MT1-MMP, a membrane-bound, cell surface type of MMP that has been previously associated with cancer invasion. Migration and the presence of MMP2 were also correlated, but to a lesser degree. We propose that membrane-bound MMPs may focus proteolysis of Ln-5 at some basement membrane "hot spots", thus defining the leading edge of invading tumors. MMP2 may subsequently function as an amplification loop to increase the efficiency of migration. We then precisely mapped where MMP is cutting the g 2 subunit of Ln-5, by a combination of MT1-MMP or MMP2 cleavage of tagged recombinant g 2 fragments, and direct amino-acid sequencing of MMP generated g 2 fragments. By these approaches we demonstrated that MT1-MMP cleaves Ln-5 g 2 in two places, liberating a small EGF-like peptide that might have functional activity. To assess the in vivo validity of these cleavage mechanisms, we investigated tissue of mice that genetically lack MT1-MMP. We found that in several epithelial tissues, including kidney, lung and epidermis, Ln-5 cleavage is absent or greatly reduced. In the same tissue, abnormal organization of epithelial layers was evident.

In summary, we have characterized MMP dependent molecular mechanisms that affect breast cancer cell migration via cleavage of Ln-5, and possible production of migratory chemical cues. This knowledge may be useful to define novel targets for blocking cell migration, and hopefully prevent or limit cancer invasion and metastasis.

Cell migration through extracellular matrix: membrane-type metalloproteinases make the way
Periodical:Journal of Cell Biology
Index Medicus: J Cell Biol
Authors: Quaranta, V
Yr: 2000 Vol: 149 Nbr: 6 Abs: Pg:1167-70

Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7
Periodical:Cell Communication and Adhesion
Index Medicus: Cell Commun Adhes
Authors: Kiosses WB, Hahn KM, Giannelli G, Quaranta V
Yr: 2001 Vol: 8 Nbr: 1 Abs: Pg:29-44

Inhibitory role of alpha 6 beta 4-associated erbB-2 and phosphoinositide 3-kinase in keratinocyte haptotactic migration dependent on alpha 3 beta 1 integrin
Periodical:Journal of Cell Biology
Index Medicus: J Cell Biol
Authors: Hintermann E, Bilban M, Sharabi A, Quaranta V
Yr: 2001 Vol: 153 Nbr: 3 Abs: Pg:465-78