Kiyohito Mizutani

Localization of nectin-free afadin at the leading edge and its involvement in directional cell movement induced by platelet-derived growth factor

Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, αvβ3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.

Interaction of Nectin-like Molecule 2 with Integrin α6β4 and Inhibition of Disassembly of Integrin α6β4 from Hemidesmosomes

In normal epithelial cells, integrin α6β4 is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α6β4 is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α6β4. The binding of Necl-2 with integrin β4 was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α6β4 was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α6β4 complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.

Reduction of the ST6 β-Galactosamide α-2,6-Sialyltransferase 1 (ST6GAL1)-catalyzed Sialylation of Nectin-like Molecule 2/Cell Adhesion Molecule 1 and Enhancement of ErbB2/ErbB3 Signaling by MicroRNA-199a

Background: Nectin-like molecule 2 (Necl-2) is a tumor suppressor and suppresses ErbB2/ErbB3 signaling. Results: MicroRNA-199a (miR-199a) targets the glycosylation enzyme ST6GAL1, reduces the sialylation of Necl-2, indirectly reduces the protein level of Necl-2, and enhances ErbB2/ErbB3 signaling. Conclusion: miR-199a indirectly regulates Necl-2 and enhances ErbB2/ErbB3 signaling. Significance: MicroRNAs regulate the glycosylation of plasma membrane proteins. Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30–60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 β-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Interaction of Necl‐4/CADM4 with ErbB3 and integrin α6β4 and inhibition of ErbB2/ErbB3 signaling and hemidesmosome disassembly

Nectin‐like molecule 4 (Necl‐4)/CADM4, a transmembrane cell–cell adhesion molecule with three Ig‐like domains, was shown to serve as a tumor suppressor, but its mode of action has not been elucidated. In this study, we showed that Necl‐4 interacted in cis with ErbB3 through their extracellular regions, recruited PTPN13 and inhibited the heregulin‐induced activation of the ErbB2/ErbB3 signaling. In addition, we extended our previous finding that Necl‐4 interacts in cis with integrin α6β4 through their extracellular regions and found that Necl‐4 inhibited the phorbol ester‐induced disassembly of hemidesmosomes. These results indicate that Necl‐4 serves as a tumor suppressor by inhibiting the ErbB2/ErbB3 signaling and hemidesmosome disassembly.

Absence of primary cilia in cell cycle-arrested human breast cancer cells

Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki‐67, primary cilia were present only in 20–40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indicate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells.

Necl-5/PVR enhances PDGF-induced attraction of growing microtubules to the plasma membrane of the leading edge of moving NIH3T3 cells.

Microtubules (MTs) search for and grow toward the leading edge of moving cells, followed by their stabilization at a specific structure at the rear site of the leading edge. This dynamic re‐orientation of MTs is critical to directional cell movement. We previously showed that Necl‐5/poliovirus receptor (PVR) interacts with platelet‐derived growth factor (PDGF) receptor and integrin αvβ3 at the leading edge of moving NIH3T3 cells, resulting in an enhancement of their directional movement. We studied here the role of Necl‐5 in the PDGF‐induced attraction of growing MTs to the leading edge of NIH3T3 cells. Necl‐5 enhanced the PDGF‐induced growth of MTs and attracted them near to the plasma membrane of the leading edge of NIH3T3 cells in an integrin αvβ3‐dependent manner. Furthermore, Necl‐5 enhanced the PDGF‐induced attraction of the plus‐end‐tracking proteins (+TIPs), including EB1, CLIP170, an intermediate chain subunit of cytoplasmic dynein, and p150Glued, a subunit of dynactin, near to the plasma membrane of the leading edge. Thus, Necl‐5 plays a role in the attraction of growing MTs to the plasma membrane of the leading edge of moving cells.

miR-214 and hypoxia down-regulate Necl-2/CADM1 and enhance ErbB2/ErbB3 signaling

Necl‐2/CADM1 is down‐regulated by the promoter hypermethylation and/or the loss of heterozygosity at chromosome 11q23.2 in many types of cancers and serves as a tumor suppressor by interacting in cis with ErbB3 and suppressing the ligand‐induced ErbB2/ErbB3 signaling for cell movement and death. However, the incidence of these epigenetic and genetic abnormalities of Necl‐2 is 30–60% in these cancers. We investigated here other mechanisms that down‐regulate Necl‐2. miR‐214, that is frequently up‐regulated in a variety of cancers, targeted the 3′UTR of the Necl‐2 mRNA directly, suppressed the translation of Necl‐2 and enhanced the ligand‐induced ErbB2/ErbB3 signaling in human colon cancer Caco‐2 cells. Hypoxia reduced the Necl‐2 protein level in a manner independent of miR‐214 or hypoxia‐inducible factor‐1α in Caco‐2 cells. These results indicate that miR‐214 and hypoxia are novel regulators that down‐regulate Necl‐2 and enhance ErbB2/ErbB3 signaling.

The Cell Adhesion Molecule Necl-4/CADM4 Serves as a Novel Regulator for Contact Inhibition of Cell Movement and Proliferation

Contact inhibition of cell movement and proliferation is critical for proper organogenesis and tissue remodeling. We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells. When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell–cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor. This interaction inhibited the tyrosine-phosphorylation of the VEGF receptor through protein-tyrosine phosphatase, non-receptor type 13 (PTPN13), eventually reducing cell movement and proliferation. When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement. Necl-4 further facilitated the activation of extracellular signal-regulated kinase 1/2, eventually enhancing cell proliferation. Thus, Necl-4 serves as a novel regulator for contact inhibition of cell movement and proliferation cooperatively with the VEGF receptor and PTPN13.

Interaction of integrin α6β4 with ErbB3 and implication in heregulin-induced ErbB3/ErbB2-mediated DNA synthesis

Integrin α6β4 is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of cell–extracellular matrix junctions. In many types of cancer cells, integrin α6β4 is up‐regulated, laminin, an integrin α6β4‐binding extracellular matrix protein, is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion. It was previously shown that integrin α6β4 interacts with ErbB1 and ErbB2 and enhances cell proliferation and motility. Here we show that integrin α6β4 interacts with ErbB3 but not with ErbB1, ErbB2 or ErbB4, and enhances the heregulin‐induced, ErbB3/ErbB2 heterodimer‐mediated DNA synthesis, but not cell motility, in A549 cells.

Suppression of the TGF-β1-induced protein expression of SNAI1 and N-cadherin by miR-199a

MicroRNA miR‐199a is clustered with miR‐214 on chromosome 1 and its expression is up‐regulated by various factors that are associated with epithelial‐to‐mesenchymal transition (EMT), such as a transcriptional repressor Twist1 and transforming growth factor (TGF)‐β. miR‐199a is either up‐regulated or down‐regulated in a variety of cancers, although EMT is associated with the progression of cancer. We found here that miR‐199a suppressed the translation of SNAI1, a transcriptional repressor that plays a role in EMT, by targeting the sequence within the 3′UTR of the SNAI1 mRNA, and reduced the protein level of SNAI1. miR‐199a increased the protein level of claudin‐1 in both the TGF‐β1‐treated and ‐untreated cells at least partly by decreasing the protein level of SNAI1, a transcriptional repressor for claudin‐1. In addition, miR‐199a targeted the sequence within the 3′UTR of the N‐cadherin mRNA and suppressed the TGF‐β1‐induced increase in the protein level of N‐cadherin in a manner independent of SNAI1. These results indicate that miR‐199a suppresses the TGF‐β1‐induced protein expression of SNAI1 and N‐cadherin.