Summary Cyclophilin A (CypA) is over-expressed in a number of human cancer types, but the mechanisms by which CypA promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds to and prevents the CrkII adaptor protein from switching to the inhibited state. CrkII is involved in cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII, by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binging site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical, and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly up-regulated.
Epithelial cell-cell contact stimulates actin cytoskeleton remodeling to down-regulate branched filament polymerization-driven lamellar protrusion and subsequently to assemble linear actin filaments required for E-cadherin anchoring during adherens junction complex assembly. In this manuscript, we demonstrate that de novo protein synthesis, the β-actin 3 ′ UTR, and the β-actin mRNA zipcode are required for epithelial adherens junction complex assembly but not maintenance. Specifically, we demonstrate that perturbing cell-cell contact-localized β-actin monomer synthesis causes epithelial adherens junction assembly defects. Consequently, inhibiting β-actin mRNA zipcode/ZBP1 interactions with β-actin mRNA zipcode antisense oligonucleotides, to intentionally delocalize β-actin monomer synthesis, is sufficient to perturb adherens junction assembly following epithelial cell-cell contact. Additionally, we demonstrate active RhoA, the signal required to drive zipcode-mediated β-actin mRNA targeting, is localized at epithelial cell-cell contact sites in a β-actin mRNA zipcode-dependent manner. Moreover, chemically inhibiting Src kinase activity prevents the local stimulation of β-actin monomer synthesis at cell-cell contact sites while inhibiting epithelial adherens junction assembly. Together, these data demonstrate that epithelial cell-cell contact stimulates β-actin mRNA zipcode-mediated monomer synthesis to spatially regulate actin filament remodeling, thereby controlling adherens junction assembly to modulate cell and tissue adhesion.
Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized β-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and β-actin translation dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed β-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3′UTR) in MDCK cells to perturb actin filament remodeling and anchoring and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated β-actin translation and dynamin mediated endocytosis regulates epithelial monolayer structure and barrier function.
The Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex at the cell surface of prostate cancer (PCa) cells influences cell–cell cohesion and dyscohesion. We investigated matrix metalloproteinase-7/matrilysin (MMP-7)’s ability to digest components of the PSPN Complex in bone metastatic PCa cells using in silico analyses and in vitro experiments. Results demonstrated that in addition to the heparan sulfate proteoglycan, perlecan, all components of the PSPN Complex were degraded by MMP-7. To investigate the functional consequences of PSPN Complex cleavage, we developed a preformed microtumor model to examine initiation of cell dispersion after MMP-7 digestion. We found that while perlecan fully decorated with glycosaminoglycan limited dispersion of PCa microtumors, MMP-7 initiated rapid dyscohesion and migration even with perlecan present. Additionally, we found that a bioactive peptide (PLN4) found in perlecan domain IV in a region subject to digestion by MMP-7 further enhanced cell dispersion along with MMP-7. We found that digestion of the PSPN Complex with MMP-7 destabilized cell–cell junctions in microtumors evidenced by loss of co-registration of E-cadherin and F-actin. We conclude that MMP-7 plays a key functional role in PCa cell transition from a cohesive, indolent phenotype to a dyscohesive, migratory phenotype favoring production of circulating tumor cells and metastasis to bone.
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