Cell adhesion and migration contribute to normal processes such as differentiation, embryonic development, and wound healing as well as to the progression of diseases and pathological conditions that can result from either acute or chronic exposure to environmental toxicants, such as cancer and inflammatory responses. Key mechanistic steps in these processes involve the interactions of extracellular glycoproteins--such as fibronectin, laminin, and collagens--with specific adhesive receptors, the best characterized of which are the integrins, a family of heterodimeric complexes consisting of an alpha subunit and a beta subunit. Integrins are highly regulated receptors that can exist in either an active or inactive state. Selectins are transemembrane cell-cell adhesion glycoproteins involved in leukocyte trafficking, inflammation, thrombosis, autoimmunity and cancer. Accumulation of leukocytes at sites of inflammation is initiated by selectins that mediate the capturing and rolling of leukocytes on endothelium. Three major members of the selectin family have been identified: L-selectin, E-selectin and P-selectin. L-Selectin is constitutively expressed on leukocytes. P-and E-selectins are expressed on activated endothelial cells in response to microenvirnomental stimuli. P-selectin is expressed on activated platelets as well as endothelial cells as a result of a secretory cascade initiated in response to environmental factors that result in inflammation. In some diseases, including certain cancers, elevated levels of P-selectin can be found in the blood. All three members of the selectin family, E-, L-, and P-selectin can bind to human tumor cells and cancer-derived cell lines. Our research has focused recently on the possible role of cues from the tumor microenvironment that can regulate integrin-mediated tumor cell migration and invasion. As a model system, we are examining the ability of one selectin, P-selectin, to trigger integrin-mediated adhesion and migration of cultured human tumor cells. We focus on two closely related aspects of this project: to characterize the mechanisms of P-selectin-induced activation of integrin-mediated cell adhesion and cell migration. We have previously shown that binding of soluble, recombinant P-selectin-IgG Fc chimeric protein to Colo-320 cells stimulates cell adhesion to fibronectin through the specific activation of the alpha5-beta1 integrin by means of the p38 MAP kinase and PI-3 kinase (PI3-k) signaling pathways. We have identified nucleolin as a novel cell surface P-selectin receptor on Colo-320 cells using affinity chromatography and a proteomic approach and that P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell surface nucleolin, PI 3-K, and p38 MAPK. We have recently been characterizing the stimulation of integrin-mediated tumor cell migration by the binding of soluble, recombinant P-selectin-IgG Fc chimeric protein. We have found that P-selectin binds to cultured human A375 melanoma cells in a concentration-dependent and saturable manner via cell surface nucleolin resulting in concommitant stimulation of matrix metalloproteinase-9 (MMP-9) secretion. We are currently investigating the mechanism by which P-selectin binding to A375 melanoma cells stimulates MMP-9 secretion. Our preliminary data indicate that P-selectin stimulates a 3-fold increase of MMP-9 mRNA, as judged by RT-PCR. Activation of p38 MAP kinase may be involved in this process. The current paradigm in the field states that MMP-9 stimulates migration by digesting basement membrane proteins, allowing tumor cells to clear a migratory pathway. However, we have preliminary data that show that stimulation of cell migration by MMP-9 using two indepedent in vitro migration assays can occur even in the absence of catalytic activity. In collaboration with investigators at Duke University, we have shown that exogenous expression of the tumor supressor CYLD markedly inhibits melanoma cell proliferation and migration in vitro and subcutaneous tumor growth in vivo. In addition, the melanoma cells expressing exogenous CYLD were unable to form pulmonary tumor nodules following tail-vein injection. At the molecular level, CYLD decreased beta-1 integrin expression and inhibited pJNK induction by TNF-alpha or cell-attachment to collagen IV. Moreover, CYLD induced an array of other molecular changes associated with modulation of the malignant phenotype, including a decreased expression of cyclin D1, N-cadherin and nuclear Bcl3, and an increased expression of p53 and E-cadherin. Most interestingly, co-expression of the constitutively active MKK7 or c-Jun mutants with CYLD prevented the above molecular changes, and fully restored melanoma growth and metastatic potential in vivo. Our findings demonstrate that JNK/AP-1 signaling pathway underlies the melanoma growth and metastasis that is associated with CYLD loss-of-function. Thus, restoration of CYLD and inhibition of JNK and beta-1 integrin function represent potential therapeutic strategies for treatment of malignant melanoma.
