Our results indicate an important role for melanoma cell surface heparin sulfate proteoglycans (HSPG) in mediating arginyl-glycyl-aspartyl (RGD)- independent cell adhesion to the 33kD carboxyl-terminal heparin binding fragment of plasma fibronectin (FN) A-chains. This is supported by several observations made during our initial funding period. First, HSPGs, isolated from metabolically-labelled tumor cells in vitro, bind to substrate coated with the 33kD fragment in a concentration dependent, saturable fashion. Secondly, HSPG binding to this fragment is specifically abolished by a cell adhesion-inhibiting monoclonal antibody (MAB) generated against the 33 kD fragment. Thirdly, we have identified novel cell adhesion-promoting synthetic peptides from the 33 kD fragment which also bind 3H-heparin and melanoma cell surface HSPG. Furthermore, the cell adhesion promoting activity of these synthetic peptides can be specifically inhibited by exogenous heparin and heparin sulfate glycosaminoglycans (GAGs). These peptides, termed I and II, have the primary sequences YEKPGSPPREVVPRPRPGV and KNNQKSEP-LIGRKKT, respectively (McCarthy, 1988b). We also have evidence for the involvement of an alpha 4 beta 1 integrin in cell recognition of this region of FN A-chains. This is based, in part, on recently published results (Wayner, et al, 1989) demonstrating a role for the interaction of alpha 4 beta 1 integrin with another cell adhesion promoting sequence within this fragment, termed CS1 (DELPQLVTLPHPNLHPGPEILDVPSKT; Humphries, 1987). Unlike peptides I and II, peptide CS1 fails to bind 3H-heparin and is only expressed in plasma FN A- chains. Although cross competition experiments suggest that peptides I, II and CS1 promote murine melanoma cell adhesion by distinct molecular mechanisms, our preliminary results demonstrate that specific alpha 4 beta 1 MABs also inhibit cell adhesion to heparin binding peptides I and II, suggesting that peptides I, II, and CS1 represent portions of a larger active site on intact FN A-chains which can bind both alpha 4 beta 1 integrins and cell surface HSPG. The underlying, and central, hypothesis to be tested in these studies is that these three sites, within the larger domain, drive the association of HSPG and alpha 4 beta 1 integrin on the cell surface as a consequence of melanoma cell adhesion to this region of FN A-chains. We will use specific anti-integrin and anti-HSPG MABs, in conjunction with other reagents such as synthetic peptides and restricted recombinant FN fragments containing this domain, to confirm or refute predictions stemming from this hypothesis. Specific comparisons will be made between highly metastatic melanoma cells and poorly metastatic counterparts, to attempt to identify specific changes in cell adhesion phenotype which correlate with invasive or metastatic behavior.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043924-06
Application #
2091325
Study Section
Pathology A Study Section (PTHA)
Project Start
1987-09-01
Project End
1995-02-28
Budget Start
1993-03-01
Budget End
1995-02-28
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Pathology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Ybarrondo, B; O'Rourke, A M; McCarthy, J B et al. (1997) Cytotoxic T-lymphocyte interaction with fibronectin and vitronectin: activated adhesion and cosignalling. Immunology 91:186-92
Henke, C A; Roongta, U; Mickelson, D J et al. (1996) CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix. J Clin Invest 97:2541-52
Huhtala, P; Humphries, M J; McCarthy, J B et al. (1995) Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol 129:867-79
Hansen, L K; O'Leary, J J; Skubitz, A P et al. (1995) Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70 kDa family of heat-shock proteins. Biochim Biophys Acta 1252:135-45
Iida, J; Meijne, A M; Spiro, R C et al. (1995) Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and alpha 4 beta 1 integrin. Cancer Res 55:2177-85
Braunewell, K H; Pesheva, P; McCarthy, J B et al. (1995) Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth. Eur J Neurosci 7:805-14
Giuseppetti, J M; McCarthy, J B; Letourneau, P C (1994) Isolation and partial characterization of a cell-surface heparan sulfate proteoglycan from embryonic rat spinal cord. J Neurosci Res 37:584-95
Katoh, S; McCarthy, J B; Kincade, P W (1994) Characterization of soluble CD44 in the circulation of mice. Levels are affected by immune activity and tumor growth. J Immunol 153:3440-9
Wahl, S M; Allen, J B; Hines, K L et al. (1994) Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment. J Clin Invest 94:655-62
Wilke, M S; Vespa, J; Skubitz, A P et al. (1993) Human keratinocytes adhere to and spread on synthetic peptide FN-C/H-V derived from fibronectin. J Invest Dermatol 101:43-8

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