Selectins (P-, L- and E-selectin) and fibrin(ogen) play pivotal roles in the metastatic spread of tumor cells. Selectin deficiency interferes with the initial tethering/ seeding and subsequent lodging of metastatic cells in target organs, whereas depletion of fibrin(ogen) impedes the sustained adhesion and stable implantation of tumor cells in the microvasculature. Our data revealing that the coordinated action of selectin-dependent tethering followed by integrin-mediated firm adhesion is requisite for maximal binding of colon carcinoma cells to host cells may offer a unifying perspective by which selectins and fibrin(ogen) act in concert in altering tumor cell metastatic potential. However, the selectin and fibrin(ogen) ligands on colon carcinoma cells have yet to be delineated. Sialofucosylated CD44 variant isoforms (CD44v) represent the major functional P-selectin ligands on colon carcinoma cells, and act as auxiliary L-, and possibly E-, selectin ligands. Although other selectin ligands have been identified on tumor cell lines, their functional roles and physiological significance have not been substantiated. Similarly, the fibrin(ogen) receptor on colon carcinoma remains elusive. By identifying the major functional L- and E-selectin ligand(s) and fibrin(ogen) binding molecules on colon carcinoma cells, and characterizing their biochemical and biophysical properties, we will provide the basis to engineer novel therapeutic agents that selectively block tumor cell binding to host cells, and thus interfere with metastatic spread. These studies will also identify targets for developing effective cancer cell drug-delivery therapies. The current application proposes to use human colon carcinoma cells, since colon cancer is prevalent in the United States and is among those tumors with a propensity for hematogenous spread.
In Aim 1, we will characterize the biochemical nature of CD44-fibrin interaction using flow-based adhesion assays in conjunction with highly specific enzymes and glycoconjugate biosynthesis inhibitors. Moreover, we will identify the CD44 binding site on the fibrin molecule, using biochemical and molecular biology techniques.
In Aim 2, the kinetic and micromechanical properties of the CD44-fibrin and CD44-P-selectin pairs will be determined using force spectroscopy. This technology will enable us to probe both the affinity and avidity of receptor-ligand binding under force.
In Aim 3, we will isolate the major functional L- and E-selectin ligands from colon carcinoma cells, and characterize their critical selectin binding determinants and binding kinetics, using a combination of biochemical, biophysical and molecular cell biology techniques. To assess the functional role of these ligands, stable LS174T cell lines will be generated, in which the expression of these molecules will be knocked down using short-hairpin RNA technology. The knowledge gained from these studies will bring together disparate observations about the contributions of selectins, fibrin(ogen) and CD44 in one mechanistic interpretation, and may reveal novel therapeutic approaches to impede metastasis. Project Narrative: Metastasis, which refers to the dissemination of tumor cells from a primary tissue to secondary sites in target organs, represents the most elaborate barrier to overcome in the cure of cancer. Here, we propose to identify targets for developing effective cancer cell drug-delivery therapies to impede metastasis, by employing a combination of biophysical, biochemical and molecular cell biology techniques.
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