Molecular Characterization of Cell-Cell Interactions in Cancer Metastasis

Johns Hopkins University, Baltimore MD

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Abstract

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 sustained adhesion and stable implantation of tumor cells in the microvasculature. Sialofucosylated CD44 variant isoforms (CD44v) represent the major functional P-selectin ligands on colon carcinoma cells, and act as auxiliary L- and E-selectin ligands. Although other selectin ligands have previously 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, and characterizing their biochemical and biophysical properties, we will provide the basis to engineer novel therapeutic agents that will selectively block tumor cell binding to host cells, and thus interfere with metastatic spread. The current application proposes to use human colon carcinoma cells, since colon cancer is prevalent in the United States and is also among those tumors with a propensity for hematogenous spread. In Aim 1, we will isolate and characterize the major functional L- and E-selectin ligands on colon carcinoma cells, using a combination of standard and sophisticated biochemical and molecular cell biology techniques. To assess the functional role of these ligands, stable LS174T cell lines will be generated, in which the putative molecules will be knocked down using short-hairpin RNA technology. Aim 2 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 3, the kinetic and micromechanical properties of the relevant receptor-ligand 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. The knowledge gained from these studies will bring together disparate observations about the contributions of selectins, fibrin(ogen) and CD44 in one mechanistic intepretation, and may offer novel avenues for metastatic control.