Polymerized Laminin-332 Matrix Supports Rapid and Tight Adhesion of Keratinocytes, Suppressing Cell Migration

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration

N-Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

α6ß4 integrin is an essential component of hemidesmosomes and modulates cell migration in wound healing and cancer invasion. To elucidate the role of N-glycosylation on ß4 integrin, we investigated keratinocyte adhesion and migration through the re-expression of wild-type or N-glycosylation-defective ß4 integrin (ΔNß4) in ß4 integrin null keratinocytes. N-glycosylation of ß4 integrin was not essential for the heterodimer formation of ß4 integrin with α6 integrin and its expression on a cell surface, but N-glycosylation was required for integrin-mediated cell adhesion and migration. Concomitantly with the reduction of ß4 integrin in the membrane microdomain, the intracellular signals of Akt and ERK activation were decreased in cells expressing ΔNß4 integrin. Forced cross-linking of ß4 integrin rescued the decreased ERK activation in ΔNß4 integrin-expressing cells to a similar extent in wild-type ß4 integrin-expressing cells. Surprisingly, compared with cells expressing wild-type ß4 integrin, an alternation in N-glycan structures expressed on epidermal growth factor receptor (EGFR), and the induction of a stronger association between EGFR and ß4 integrin were observed in ΔNß4 integrin-expressing cells. These results clearly demonstrated that N-glycosylation on ß4 integrin plays an essential role in keratinocyte cellular function by allowing the appropriate complex formation on cell surfaces

Quantitative analysis of β1,6GlcNAc-branched N-glycans on β4 integrin in cutaneous squamous cell carcinoma

α6β4 integrin plays pivotal roles in cancer progression in several types of cancers. Our previous study using N-glycan-manipulated cell lines demonstrated that defects in N-glycans or decreased β1,6GlcNAc-branched N-glycans on β4 integrin suppress β4 integrin-mediated cancer cell adhesion, migration, invasion, and tumorigenesis. Furthermore, immunohistochemical analysis has shown that colocalization of β1,6GlcNAc-branched N-glycans with β4 integrin was observed in cutaneous squamous cell carcinoma (SCC) tissue. However, until now there has been no direct evidence that β1,6GlcNAc-branched N-glycans are upregulated on β4 integrin in cutaneous SCC. In the present study, we performed an ELISA analysis of β1,6GlcNAc-branched N-glycans on β4 integrins as well as β4 integrins in cell lysates from human normal skin and cutaneous SCC tissues. The SCC samples showed a 4.9- to 7.4-fold increase in the ratio of β1,6GlcNAc-branched N-glycans to β4 integrin compared with normal skin samples. These findings suggest that the addition of β1,6GlcNAc-branched N-glycans onto β4 integrin was markedly elevated in cutaneous SCC tissue compared to normal skin tissue. The value of β1,6GlcNAc-branched N-glycans on β4 integrin may be useful as a diagnostic marker associated with cutaneous SCC tumor progression

Osteopontin in Cancer: Mechanisms and Therapeutic Targets

Despite significant advances in the understanding of cancer biology, cancer is still a leading cause of death worldwide. Expression of the tumor microenvironment component, osteopontin, in tumor tissues, plasma, and serum, has been shown to be associated with a poor prognosis and survival rate in various human cancers. Recent studies suggest that osteopontin drives tumor development and aggressiveness using various strategies. In this review, we first provide an overview of how osteopontin promotes tumor progression, such as tumor growth, invasion, angiogenesis, and immune modulation, as well as metastasis and chemoresistance. Next, we address how the functional activities of osteopontin are modulated by the interaction with integrins and CD44 receptors, but also by the post-translational modification, such as proteolytic processing by several proteases, phosphorylation, and glycosylation. Then, we review how osteopontin activates tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), and functions as an immunosuppressor by regulating immune surveillance and immune checkpoint in the tumor microenvironment. Finally, we discuss the potential applications of osteopontin as a biomarker and as a therapeutic target

Osteopontin in Cancer: Mechanisms and Therapeutic Targets

Despite significant advances in the understanding of cancer biology, cancer is still a leading cause of death worldwide. Expression of the tumor microenvironment component, osteopontin, in tumor tissues, plasma, and serum, has been shown to be associated with a poor prognosis and survival rate in various human cancers. Recent studies suggest that osteopontin drives tumor development and aggressiveness using various strategies. In this review, we first provide an overview of how osteopontin promotes tumor progression, such as tumor growth, invasion, angiogenesis, and immune modulation, as well as metastasis and chemoresistance. Next, we address how the functional activities of osteopontin are modulated by the interaction with integrins and CD44 receptors, but also by the post-translational modification, such as proteolytic processing by several proteases, phosphorylation, and glycosylation. Then, we review how osteopontin activates tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), and functions as an immunosuppressor by regulating immune surveillance and immune checkpoint in the tumor microenvironment. Finally, we discuss the potential applications of osteopontin as a biomarker and as a therapeutic target

Roles of Integrin α6β4 Glycosylation in Cancer

Malignant transformation is accompanied with aberrant glycosylation of proteins. Such changes in glycan structure also occur in the integrins, which are a large family of cell surface receptors for the extracellular matrix and play key roles in tumor progression. There is now increasing evidence that glycosylation of integrins affects cellular signaling and interaction with the extracellular matrix, receptor tyrosine kinases, and galectins, thereby regulating cell adhesion, motility, growth, and survival. Integrin α6β4 is a receptor for laminin-332 and the increased expression level is correlated with malignant progression and poor survival in various types of cancers. Recent studies have revealed that integrin α6β4 plays central roles in tumorigenesis and the metastatic process. In this review, we summarize our current understanding of the molecular mechanisms of tumor progression driven by integrin α6β4 and also discuss the modification of glycans on integrin β4 subunit to address the important roles of glycan in integrin-mediated tumor progression

Roles of Integrin α6β4 Glycosylation in Cancer

Malignant transformation is accompanied with aberrant glycosylation of proteins. Such changes in glycan structure also occur in the integrins, which are a large family of cell surface receptors for the extracellular matrix and play key roles in tumor progression. There is now increasing evidence that glycosylation of integrins affects cellular signaling and interaction with the extracellular matrix, receptor tyrosine kinases, and galectins, thereby regulating cell adhesion, motility, growth, and survival. Integrin α6β4 is a receptor for laminin-332 and the increased expression level is correlated with malignant progression and poor survival in various types of cancers. Recent studies have revealed that integrin α6β4 plays central roles in tumorigenesis and the metastatic process. In this review, we summarize our current understanding of the molecular mechanisms of tumor progression driven by integrin α6β4 and also discuss the modification of glycans on integrin β4 subunit to address the important roles of glycan in integrin-mediated tumor progression

Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility

Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility

Immunofluorescent staining of Lm332 deposited by three cell lines.

<p>NHK (A, top), HSC-4 cells (A, center) and Lm332-HEK cells (A. bottom) were suspended in serum-free medium, inoculated at a cell density of 2×10³ cells/well on collagen-coated 8-well chamber slides and incubated for 6 h. The cultures were stained for F-actin with rhodamine phalloidin (left panels) and for Lm332 with the anti-α3 chain BG5 antibody and followed by a FITC-labeled secondary antibody (center panels), as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035546#s4" target="_blank">Materials and Methods</a>”. Right panels are merged images. In (B), Lm332-HEK cells were inoculated at a high cell density (1×10⁵ cells/well), incubated for 6 h (left panel) or 48 h (right panel), and stained for Lm332 as above.</p