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Cell Migration Across Basement Membranes in Tissue Remodeling and Metastasis

V. Quaranta, M. Bilban, E. Hintermann, N. Koshikawa, L. Nisi,* T.A. Romano, S. Schenk, M. Shang, A. Sharabi, N. Yang, D. Richards**

* University of Bari Medical School, Bari, Italy
** University of California, San Francisco, CA

Invading neighboring tissue structures, spreading throughout the body, and forming distant sites of metastatic growth are the lethal properties of cancer. Critical to such properties is the switching of cancer cells to a mobile state, which reflects the migratory state of normal cell types in tissues undergoing remodeling or repair. We hypothesize that in cancer cells, overactive migration-inducing mechanisms cause escape from primary sites, leading to local invasion and metastasis. Several projects are designed to test this hypothesis. Our data support the notion that interactions among macromolecules of the extracellular matrix, integrin adhesion receptors, and matrix metalloproteinases (MMPs) play a primary role in the migration of cancer cells. Understanding these interactions may provide means for preventing or limiting cancer invasion and metastasis.

INTEGRINS IN LAMININ-5 MIGRATION

In epithelia, from which most human cancers originate, the extracellular matrix is condensed in specialized mats called basement membranes, on which epithelial cells rest. Within basement membranes, a family of molecules, the laminins, provide an interface between epithelial cells and underlying tissue and modulate several cellular functions, including migration. One focus in our research is the role of the isoform laminin-5 in cancer invasion. This laminin is found in the basement membranes of tissues from which tumors often arise (e.g., lung, prostate, mammary gland, gastrointestinal tract).

Two integrins, a₃ß₁ and a₆ß₄, are the epithelial cell receptors for laminin-5. In remodeling or healing epidermis, a₃ß₁ is located at the leading edge of migrating keratinocytes. In contrast, a₆ß₄ anchors quiescent epithelia to the underlying basement membrane via specialized structures termed hemidesmosomes. We hypothesize that a₃ß₁ implements migration, and a₆ß₄ implements epithelial static adhesion. To test this hypothesis, we are investigating signaling interactions between a₃ß₁ and a₆ß₄.

In keratinocytes, we distinguished 2 types of migration on laminin-5. One type, haptotactic migration, depends strictly on interactions between integrins and laminin-5 and is triggered by activated a₃ß₁. The other, chemotactic migration, is stimulated by growth factors such as epidermal growth factor and although a₃ß₁-dependent does not require activation of this integrin. Another significant difference is that haptotactic, but not chemotactic, migration is sensitive to inhibition by a₆ß₄; such inhibition may occur when a₆ß₄ is engaged with ligand. Inhibition of a₃ß₁ migration is mediated by a₆ß₄-associated erbB-2, via phosphatidylinositol-3´-kinase. Haptotactic keratinocyte migration may reflect late stages of tissue repair, when a₆ß₄ may exert a negative influence on the migratory a₃ß₁ in order to anchor newly quiescent epidermis. A role for this molecular signaling circuit in cancer invasion should be investigated.

METALLOPROTEINASES AND MIGRATION ON LAMININ-5

We previously found that MMPs can turn laminin-5 into a migratory substrate by cleaving its g₂ subunit. It is now increasingly clear that MT1-MMP, a membrane-bound metalloproteinase, is the main enzyme that cleaves laminin-5. In mice that lack the gene for MT1-MMP, kindly provided by our collaborators, H. Birkedal-Hansen, K. Holmbeck, and P. Bianco, National Institute of Dental and Craniofacial Research, Bethesda, Maryland, no cleavage of laminin-5 occurs. In collaboration with R. Zent, Vanderbilt University, Nashville, Tennessee, we are investigating possible phenotypes of epithelial organization. Another metalloproteinase, MMP2, may play an amplification role in proteolysis of laminin-5 g₂ subunits, resulting in fragments that may bind to motility-stimulating cell-surface receptors. We now need to define the role in cancer invasion of MMP2, MT1-MMP, and laminin-5, particularly the g₂ subunit, because these molecules are sometimes found at the leading edges of invasive cancers.

In a related project, we are investigating whether MMPs and laminin-5 are involved in trophoblast invasion. In these studies on "normal" invasive processes, which nicely complement cancer studies, we are using DNA microarray technology to profile expression of invasion- and migration-related genes in invasive versus quiescent trophoblasts.

THE BINDING SITE FOR a₃ß₁ ON LAMININ-5

We found that a small laminin-5 a₃ subunit domain, LG3, is the binding site for integrin a₃ß₁. Recombinant LG3 supports a₃ß₁-dependent adhesion and migration; specifically binds a₃ß₁, as indicated by affinity chromatography; and stimulates phosphorylation of focal adhesion kinase. The LG3 recombinant protein is a unique, defined reagent for dissecting structural and functional properties of cell adhesion and migration onto laminin-5.

EPITHELIAL CELL MIGRATION AT THE TOOTH-GINGIVAL INTERFACE

In collaboration with S. Kinder-Haake, University of California, Los Angeles, we are continuing our studies on laminin-5 at the tooth-gum interface. Our initial results indicate that migration of oral epithelial cells on laminin-5 may be modified by periodontal bacteria. We will attempt to clarify the biochemical basis for this phenomenon.

PUBLICATIONS
Bilban, M., Head, S., Desoye, G., Quaranta, V. DNA microarrays: A novel approach to investigate genomics in trophoblast invasion--a review. Placenta 21(Suppl. A):S99, 2000.

Cirulli, V., Beattie, G.M., Klier, G., Ellisman, M., Ricordi, C., Quaranta, V., Frasier, F., Ishii, J.K., Hayek, A., Salomon, D.R. Expression and function of a_vß₃ and a_vß₅ integrins in the developing pancreas: Roles in the adhesion and migration of putative endocrine progenitor cells. J. Cell Biol. 150:1445, 2000.

Giannelli, G., Bergamini, C., Fransvea, E., Marinosci, F., Quaranta, V., Antonaci, S. Human hepatocellular carcinoma (HCC) cells require both a₃ß₁ integrin and matrix metalloproteinases activity for migration and invasion. Lab. Invest. 81:613, 2001.

Hintermann, E., Bilban, M., Sharabi, A., Quaranta, V. Inhibitory role of a₆ß₄-associated erbB-2 and phosphoinositide 3-kinase in keratinocyte haptotactic migration dependent on a₃ß₁ integrin. J. Cell Biol. 153:465, 2001.

Koshikawa, N., Giannelli, G., Cirulli, V., Miyazaki, K., Quaranta, V. Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5. J. Cell Biol. 148:615, 2000.

Plopper, G.E., Huff, J.L., Rust, W.L., Schwartz, M.A., Quaranta, V. Antibody-induced activation of ß₁ integrin receptors stimulates cAMP-dependent migration of breast cells on laminin-5. Mol. Cell Biol. Res. Commun. 4:129, 2000.

Quaranta, V. Cell migration through extracellular matrix: Membrane-type metalloproteinases make the way. J. Cell Biol. 149:1167, 2000.

Seftor, R.E., Seftor, E.A., Koshikawa, N., Meltzer, P.S., Gardner, L.M., Bilban, M., Stetler-Stevenson, W.G., Quaranta, V., Hendrix, M.J. Cooperative interactions of laminin 5 g₂ chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma. Cancer Res. 61: 6322, 2001.

Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. The LG3 module of laminin-5 harbors a binding site for integrin a₃ß₁ that promotes cell adhesion, spreading and migration. J. Biol. Chem. 226:33045, 2001.

Zent, Z., Bush, T.K., Martin, L., Pohl, L.M., Quaranta, V., Koshikawa, N., Wang, Z., Kreidberg, A., Sakurai, H., Stuart, O.R., Nigam, K.S. Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development. Dev. Biol., in press.

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