Colon adenocarcinomas are known to express elevated levels of A2-6 sialylation and increased activity of ST6Gal-I, the Golgi glycosyltransferase that creates A2-6 linkages. Elevated ST6Gal-I positively correlates with metastasis and poor survival, and therefore ST6Gal-I-mediated hypersialylation likely plays a role in colorectal tumor invasion. Previously we found that oncogenic ras (present in roughly 50% of colon adenocarcinomas) up-regulates ST6Gal-I and, in turn, increases sialylation of B 1 integrin adhesion receptors in colon epithelial cells. However, we wanted to know if this pattern held true in vivo and, if so, how B 1 hypersialylation might contribute to colon tumor progression. In the present study, we find that B 1 integrins from colon adenocarcinomas consistently carry higher levels of A2-6 sialic acid. To explore the effects of increased A2-6 sialylation on B 1 -integrin function, we stably expressed ST6Gal-I in a colon epithelial cell line lacking endogenous ST6Gal-I. ST6Gal-I expressors (with A2-6 sialylated B 1 integrins) exhibited up-regulated attachment to collagen I and laminin and increased haptotactic migration toward collagen I, relative to parental cells (with completely unsialylated B 1 integrins). Blockade of ST6Gal-I expression with short interfering RNA reversed collagen binding back to the level of ST6Gal-I nonexpressors, confirming that a2-6 sialylation regulates B 1 integrin function. Finally, we show that B 1 integrins from ST6Gal-I expressors have increased association with talin, a marker for integrin activation. Collectively, these findings suggest that B 1 hypersialylation may augment colon tumor progression by altering cell preference for certain extracellular matrix milieus, as well as by stimulating cell migration. (Cancer Res 2005; 65(11): 4645-52)

Intense investigation has centered on understanding the regulation of integrin cell adhesion receptors. In the present study, we propose that variant N-glycosylation represents an important mechanism for regulation of beta1, but not beta3 or beta5 integrins. We find that expression of oncogenic ras in HD3 colonocytes causes increased alpha2-6 sialylation of beta1 integrins, whereas expression of dominant-negative ras induces decreased alpha2-6 sialylation, relative to cells with wild-type ras. In contrast, neither beta3 nor beta5 integrins are alpha2-6 sialylated, regardless of the state of ras activation. Results from RT-PCR analyses suggest that differential integrin sialylation is due to a ras-dependent alteration in the expression of ST6Gal I, the enzyme that adds alpha2-6-linked sialic acids. Cells that express differentially sialylated beta1 integrins exhibit altered adhesion to collagen I (a beta1 ligand), but not to vitronectin (a beta3 or beta5 ligand). Similarly, the enzymatic removal of cell surface sialic acids from control cells alters binding to collagen, but not to vitronectin. Finally, using a cell-free receptor/ligand-binding assay, we show that purified, desialylated alpha1beta1 integrins have diminished collagen-binding capability, providing strong evidence that sialic acids play a causal role in regulating beta1 integrin function.

Despite numerous reports suggesting that ␤ 1 integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that ␤ 1 integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the ␤ 1 integrin subunit becomes hyposialylated, suggesting that the ␤ 1 integrin is a substrate for this enzyme. The expression of hyposialylated ␤ 1 integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified ␣ 5 ␤ 1 integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated ␤ 1 integrins are more adhesive to fibronectin, although desialylation of ␣ 5 subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the ␤ 1 integrin family of cell adhesion receptors.

Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the ␤1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of ␣5␤1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylationdependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified ␣5␤1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the ␤1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the ␤1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.The U937 and THP-1 cell lines represent well accepted model systems for studying myeloid differentiation along the monocyte/macrophage lineage. Following treatment with phorbol myristate acetate (PMA), 4 these cells exhibit phenotypic changes that are characteristic of cell differentiation, including increased respiratory burst activity, enhanced phagocytotic capability, and markedly elevated cell adhesiveness to extracellular matrix ligands such as fibronectin. In vivo, the increased adhesiveness of monocytes/macrophages contributes to the extravasation of cells from the vasculature as well as tethering of cells within inflamed tissues.Differentiating myeloid cells bind to fibronectin through the integrin family of cell adhesion receptors, including the ␣5␤1 integrin species. The molecular mechanisms underlying PMA-dependent cell adhesion have not been well defined, although it has been reported that PMA increases the synthesis of both ␣5 and ␤1 integrin subunits (1-4). However, myeloid cells (U937 and THP-1) express an abundant amount of ␣5␤1 in the absence of PMA treatment, and yet these cells bind very poorly to fibronectin. This suggests that myeloid ␣5␤1 receptors are normally in an inactive state and that increased expression alone cannot account for the dramatically increased fibronectin binding induced by PMA.In our prior study (5) we observed that PMA stimulated a rapid but transient increase in fibronectin binding that was likely due to the activation of integrins already present on the cell surface. However, following this initial transient event there was a second phase of elevated fibronect...

The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen-I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen-I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.