In this study, we have used an in vitro model system consisting of cultured Colo 320 human carcinoma cells incubated with soluble P-selectin-IgG chimeric protein to characterize the regulation by selectins of integrin function in cancer cells. We have shown that P-selectin binding to Colo 320 cells can specifically activate the α5β1 integrin, which results in increased cell attachment and cell spreading specifically on fibronectin in a concentration- and time-dependent manner. This selectin-mediated integrin activation results from at least two distinct intracellular signaling pathways—the p38 MAPK pathway and the PI3-K pathway—that are linked by a p38 MAPK-PI3-K signaling complex.
During the hematogenous phase of cancer metastasis, cells that reach the blood stream must eventually gain the ability to attach firmly to the blood vessel endothelium prior to transendothelial migration and colonization of host organs [1
]. The selectins and integrins have both been shown to play roles in this process. Selectins have been suggested to participate in the initial tethering of circulating tumor cells to host organ endothelium, probably due to abnormal glycosylation of tumor cell surface proteins can result in expression of carbohydrate moieties that mimic ligands for selectins [55
]. We observed that Colo 320 cells express at least two glycoproteins reported to function as P-selectin ligands -- CD24 and CD44 (Reyes and Akiyama, unpublished data), but we have not yet determined the role of these molecules as P-selectin ligands.
All three members of the selectin family have been shown to bind to certain transformed human cells [4
]. E-Selectin has been the most intensely studied because it was the first member of this family found to mediate adhesion of colon cancer cells to vascular endothelium. The role of L-selectin in the metastatic process has remained less clear. Current experimental evidence suggests that P-selectin can promote initial attachment of cancer cells to the blood vessel endothelium and can mediate the interactions between cancer cells and platelets to form emboli, both of which facilitate the arrest of cancer cells [2
We present several lines of evidence suggesting that P-selectin may specifically activate the α5β1 integrin on Colo 320 cells. Binding of P-selectin induced the expression of an activation-dependent epitope of β1 integrin, as judged by increased binding of the activation-specific HUTS-21 mAb (40), but did not appear to affect total β1 integrin expression on the cell surface. Similarly, we found no evidence for integrin clustering (Reyes, Styslinger, and Akiyama, unpublished data) nor can we observe any direct interaction of integrins with P-selectin (Reyes and Akiyama, unpublished data). Thus, we propose that selectin-induced integrin activation occurs by a mechanism involving a conformational change of β1 integrins in the absence of observable clustering.
We have observed that P-selectin binding induces a significant increase of adhesion of Colo 320 cells specifically to fibronectin without significantly affecting adhesion to collagen type I, collagen type lV, or laminin. Experiments with function blocking antibodies indicate that the increase in cell adhesion can be completely accounted for by the α5β1 integrin. Although our results cannot completely rule out participation of other fibronectin-binding integrins (e.g., the αvβ1 and αvβ6 integrins), the complete inhibition by either the β1 or α5 blocking antibody suggests that it is highly unlikely that integrins complexes other than the α5β1 play a meaningful role in Pselectin-mediated activation of cell adhesion to fibronectin.
Fibronectin receptors are associated with a tumorigenic phenotype and are important for metatasis [63
]. Several integrins have been reported to function as fibronectin receptors (α3β1, α4β1, α5β1, α8β1, αvβ1, αvβ3, and αvβ6) [9
]. Among these, the α5β1 integrin is generally regarded as the classical fibronectin receptor due to its ligand specificity. Normal colon has been reported to express α3β1, α5β1, and αvβ1 as fibronectin receptors but do not express αvβ3 or αvβ6 integrins [66
]. Tumor cells display altered constitutive expression patterns of integrins that bind to fibronectin compared to normal cells [69
] . Colon tumor samples and several colon carcinoma cells can present an elevated expression of the αvβ6 integrin [66
]. The elevated expression αvβ6 integrin has been associated with a significantly reduced survival time of patients with colon cancer in comparison with tumors with no or low αvβ6 expression [67
] A significant loss of tissue staining for α5β1 and α3β1 has also been observed in colon cancer, suggesting that αvβ6 integrin may be a major fibronectin-binding receptor for colon cancer cells. However, in a systematic study of integrin expression in normal colon, adenomas, and carcinomas within the same patients, it was observed that the expression of α5β1 integrin was not modified in 25% of the colon carcinomas analyzed [68
]. It will be important to examine whether P-selectin binding can modulate the function of β3 and β6 integrins in the future.
The role of the α5β1 integrin during colon cancer progression in vivo is unclear. Some reports suggest that loss of α5β1 expression could be correlated with an increase of malignancy in colon cancer. However, it has been shown that the loss of fibronectin or α5β1 integrin does not contribute to tumorigenesis or metastasis [73
]. Other reports have suggested that α5β1 integrin expression could contribute to malignant progression in colon carcinoma [74
]. Our observation that P-selectin could activate α5β1 integrin-mediated adhesion suggests that α5β1 integrin could contribute to malignant progression, consistent with a previous study showing that inhibition of the α5β1 integrin with monoclonal antibodies inhibited the experimental metastasis of MDA-MB-231 cells in athymic nude mice [76
The ability of selectins and/or selectin ligands to regulate integrin-mediated cell-adhesive events has previously been observed in leukocytes. Cross-linking of selectins induces signals that can regulate activation of β2 integrins [77
]. Integrin activation can also be triggered by selectin ligands. The E-selectin ligand, endothelial-leukocyte molecule-1, was reported to stimulate αMβ2 integrin binding to C3bi-coated erythrocytes and β2 integrin-mediated binding to endothelial cells [79
]. Similarly, the ligation of P-selectin glycoprotein ligand-1 to P-selectin was shown to activate β2 integrin-mediated cell attachment to intracellular adhesion molecule 1 [30
]. These studies suggest that selectin ligands can induce intracellular signaling to stimulate the higher-affinity β2 integrin-mediated adhesion necessary for extravasation. However, this is the first report showing evidence that P-selectin and their ligands could regulate β1 integrin-mediated adhesion and spreading by cancer cells from solid tumors, thus suggesting a mechanism by which stable tumor cell adhesion can be regulated.
Our finding that P-selectin binding to Colo 320 cells results in activation of both the PI3-K and p38 MAPK signaling pathways suggests that selectin ligands can function as signaling molecules. Both the p38 MAPK and PI3-K pathways have been previously reported to modulate integrin activity in other systems. In mast cells, PI3-K activation by FcεRI, c-kit or PDGF increases the affinity of α5β1 [81
] and in carcinoma cells, PI3-K activation by HGF increases integrin avidity [51
]. In breast cancer cells, p38 MAPK is involved in the regulation of arachidonic acid-mediated adhesion and spreading on collagen type IV [48
Interestingly, we found that only the PI3-K, and not the p38 MAPK, pathway participates in P-selectin-induced adhesion of Colo 320 cells to fibronectin. In contrast, we found both PI3-K and p38 MAPK regulate P-selectin-induced cell spreading. We also found that that a general tyrosine inhibitor, genistein, blocks completely the P-selectin-mediated PI3K activity. Since specific p38 inhibitors did not detectably inhibit PI3-K, these results suggest strongly that at least one as yet unidentified tyrosine kinase and PI3-K, but not p38 MAPK, regulate the P-selectin-induced α5β1 integrin activation. In a different system, E-selectin binding to HT-29 colon carcinoma cells has been shown to mediate transendothelial migration in a p38 MAPK-dependent manner [59
]. This result, taken together with our observations, suggests that p38 MAPK regulates more complex cell adhesion events such as cell spreading and migration, which may play a roles in cancer cell extravasation. It would be very interesting to determine whether E-selectin binding can also activate PI3-K and activate integrin-mediated adhesiveness of HT-29 cells.
We initially expected that PI 3-K would be upstream from p38 MAPK activation because the latter was not required for increased cell adhesion to fibronectin. However, a PI3-K inhibitor had no significant effect on p38 MAPK activation. Conversely, a p38 MAPK inhibitor did not affect P-selectin-induced PI 3-K activation. These data suggest that the activation of PI 3K and p38 MAPK may be independent events in P-selectin-mediated signaling pathway, consistent with previous results [84
We found that P-selectin stimulation causes the formation of a protein complex containing both PI3-K and activated p38 MAPK as judged by co-immunoprecitation experiments and the co-localization of both proteins at the cells periphery. The colocalization of p38 MAPK and PI3-K in intact P-selectin treated, but not untreated, cells suggest that complex formation is not an artifact of cell lysis. This is the first report, to our knowledge, demonstrating the formation of a specific, stimulus-dependent PI3-K-p38 signaling complex, and suggests a mechanism for crosstalk between PI3-K and p38 MAPK pathways.
Previous studies have shown that multiple stimuli can activate both the PI3-K and p38 MAPK signaling pathways leading to a variety of outcomes. In breast cancer cells, heregulin-mediated-PI3K and -p38 MAPK activation has been reported to regulate the activation the matrix metalloproteinase-2 (MMP-2) [84
] and MMP-9 [88
], proteins that play important roles in cancer cell invasion [85
]. Insulin-like growth factor can activate PI3K and p38 MAPK leading to increased breast cancer migration [89
]. Both pathways are activated in a Src family kinase-dependent manner via the epidermal growth factor (EGF) receptor and are important for EGF-mediated vascular epidermal growth factor production and promotion of the angiogenic potential of pancreatic cancer cells [86
]. Hypoxia can also activate both PI3-K and p38 MAPK pathways, which have been implicated in the regulation of IL-8 expression and angiogenesis by ovarian carcinoma cells [87
]. Out results suggest the hypothesis that formation of a P-selectin-mediated PI3-K-p38 signaling complex could lead to the specific activation of integrins as a result of P-selectin stimulation. We also propose that formation of a PI3-K-p38 signaling complex could provide a mechanism to regulate the activity of both molecules.
From analysis of protein sequences (http://www.elm.eu.org/
) and the activation mechanisms of PI3-K and p38 MAPK [90
], we suggest that the interaction between PI3-K and p38 MAPK may be through as yet unidentified adaptor proteins. Analysis of the amino acid sequences of PI3-K p85 and p38 MAPK yields possible protein binding sites —e.g.,SH2 ligand domains, SH3 ligand domains, PDZ ligands domains--that can be recognized by adaptor proteins. Interestingly, both PI3-K p85 and p38 MAPK protein sequences have functional domains that could bind to adaptor proteins such as Grb2 and the 14-3-3 protein family. Grb2 has been reported to complex with PI3K and regulate PI3K activity in several systems [94
]. The formation of a signaling complex containing p38 MAPK and Grb2 protein has not been reported. However, Grb2 has been implicated in regulation p38 MAPK activity [97
]. For example, it has been reported that the inhibition of T-cell antigen receptor-mediated Shc-Grb2 signaling complex or the decrease of Grb2 expression in mice result in a defective TCR-induced p38 MAPK activation [97
]. The 14-3-3 protein has also been reported to participate in the regulation of p38 MAPK activation [102
] and to form a signaling complex with PI3 K [105
]. Clearly it will be important to determinate other components of the PI3-K-p38 signaling complex to understand the mechnanism of P-selectin-mediated β1 integrin activation.
The role of selectins and their counter-receptors as signaling molecules in cancer cells has not been well-explored. Most studies implying selectins as signaling molecules have been carried out with leukocytes [30
]. The only selectin family member analyzed as a signaling molecule in cancer cells has been E-selectin. The binding of E-selectin to colon carcinoma HT-29 cells was reported to increase tyrosine phosphorylation of various proteins including c-Src [34
] and p38 MAPK [59
]. The role of P-selectin as a signaling molecule in cancer cells is less clear. Studies with mutant mice have shown that the absence of P-selectin results in a significant reduction in primary tumor growth and metastasis compared to P-selectin wild-type controls [60
], suggesting that P-selectin binding to cancer cells may induce activation of signals that effect cell proliferation.
An understanding of the mechanisms that regulate the attachment of circulating tumor cells to vascular endothelium of the target organ and their subsequent transendothelial migration may be clinically significant. It has already been shown that the dissociation rate for bonds between P-selectin and tumor cells is faster by an order of magnitude than between P-selectin and normal cells, such as polymorphonuclear leukocytes [110
]. Thus, selectin-mediated tethering of carcinoma cells to vascular endothelium [1
] and integrin activation is a particularly attractive step for possible therapeutic intervention for the control of metastatic disease. The identification of the role of each member of the selectin family and the identification of the P-selectin ligands that participate in the hematogenous spread of cancer could be valuable to determine whether these proteins could be used as potential therapeutic targets to inhibit cancer metastasis.