Tunicamycin as a Novel Redifferentiation Agent in Radioiodine Therapy for Anaplastic Thyroid Cancer.

The silencing of thyroid-related genes presents difficulties in radioiodine therapy for anaplastic thyroid cancers (ATCs). Tunicamycin (TM), an N-linked glycosylation inhibitor, is an anticancer drug. Herein, we investigated TM-induced restoration of responsiveness to radioiodine therapy in radioiodine refractory ATCs. 125I uptake increased in TM-treated ATC cell lines, including BHT101 and CAL62, which was inhibited by KClO4, a sodium-iodide symporter (NIS) inhibitor. TM upregulated the mRNA expression of iodide-handling genes and the protein expression of NIS. TM blocked pERK1/2 phosphorylation in both cell lines, but AKT (protein kinase B) phosphorylation was only observed in CAL62 cells. The downregulation of glucose transporter 1 protein was confirmed in TM-treated cells, with a significant reduction in 18F-fluorodeoxyglucose (FDG) uptake. A significant reduction in colony-forming ability and marked tumor growth inhibition were observed in the combination group. TM was revealed to possess a novel function as a redifferentiation inducer in ATC as it induces the restoration of iodide-handling gene expression and radioiodine avidity, thereby facilitating effective radioiodine therapy.

Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes.

We sought to evaluate the feasibility of molecular imaging using the human sodium iodide symporter (hNIS) gene as a reporter, in addition to the enhanced firefly luciferase (effluc) gene, for tracking dendritic cell (DCs) migration in living mice. A murine dendritic cell line (DC2.4) co-expressing hNIS and effluc genes (DC/NF) was established. For the DC-tracking study, mice received either parental DCs or DC/NF cells in the left or right footpad, respectively, and combined I-124 PET/CT and bioluminescence imaging (BLI) were performed. In vivo PET/CT imaging with I-124 revealed higher activity of the radiotracer in the draining popliteal lymph nodes (DPLN) of the DC/NF injection site at day 1 than DC injection site (p < 0.05). The uptake value further increased at day 4 (p < 0.005). BLI also demonstrated migration of DC/NF cells to the DPLNs at day 1 post-injection, and signals at the DPLNs were much higher at day 4. These data support the feasibility of hNIS reporter gene imaging in the tracking of DC migration to lymphoid organs in living mice. DCs expressing the NIS reporter gene could be a useful tool to optimize various strategies of cell-based immunotherapy.

TM4SF5 suppression disturbs integrin α5-related signalling and muscle development in zebrafish.

TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling.

The COOH-terminus of TM4SF5 in hepatoma cell lines regulates c-Src to form invasive protrusions via EGFR Tyr845 phosphorylation.

Transmembrane 4 L six family member 5 (TM4SF5) enhances cell migration and invasion, although how TM4SF5 mechanistically mediates these effects remains unknown. In the study, during efforts to understand TM4SF5-mediated signal transduction, TM4SF5 was shown to bind c-Src and thus hepatoma cell lines expressing TM4SF5 were analyzed for the significance of the interaction in cell invasion. The C-terminus of TM4SF5 bound both inactive c-Src that might be sequestered to certain cellular areas and active c-Src that might form invasive protrusions. Wildtype (WT) TM4SF5 expression enhanced migration and invasive protrusion formation in a c-Src-dependent manner, compared with TM4SF5-null control hepatoma cell lines. However, tailless TM4SF5(ΔC) cells were more efficient than WT TM4SF5 cells, suggesting a negative regulatory role by the C-terminus. TM4SF5 WT- or TM4SF5(ΔC)-mediated formation of invasive protrusions was dependent or independent on serum or epidermal growth factor treatment, respectively, although they both were dependent on c-Src. The c-Src activity of TM4SF5 WT- or TM4SF5(ΔC)-expressing cells correlated with enhanced Tyr845 phosphorylation of epidermal growth factor receptor. Y845F EGFR mutation abolished the TM4SF5-mediated invasive protrusions, but not c-Src phosphorylation. Our findings demonstrate that TM4SF5 modulates c-Src activity during TM4SF5-mediated invasion through a TM4SF5/c-Src/EGFR signaling pathway, differentially along the leading protrusive edges of an invasive cancer cell.

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells.

Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.

Cell adhesion-dependent serine 85 phosphorylation of paxillin modulates focal adhesion formation and haptotactic migration via association with the C-terminal tail domain of talin.

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.

Cross-talk between TGFß1 and EGFR signalling pathways induces TM4SF5 expression and epithelial-mesenchymal transition.

The EMT (epithelial-mesenchymal transition) is involved in fibrosis and cancer, and is regulated by different signalling pathways mediated through soluble factors, actin reorganization and transcription factor actions. Because the tetraspan (also called tetraspanin) TM4SF5 (transmembrane 4 L6 family member 5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that TGFß1 (transforming growth factor ß1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR [EGF (epidermal growth factor) receptor] pathway. Inhibition of EGFR activity following TGFß1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFß1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFß1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in the development of liver pathologies.

JNK signaling activity regulates cell-cell adhesions via TM4SF5-mediated p27(Kip1) phosphorylation.

Transmembrane 4L six family member 5 (TM4SF5) can regulate cell-cell adhesion and cellular morphology via cytoplasmic p27(Kip1)-mediated changes in RhoA activity. However, how TM4SF5 causes cytosolic p27(Kip1) stabilization remains unknown. In this study we found that TM4SF5-mediated Ser10 phosphorylation of p27(Kip1) required for cytosolic localization was not always correlated with Akt activity. Inhibition or suppression of c-Jun N-terminal kinase (JNK) in TM4SF5-expressing cells decreased Ser10 phosphorylation of p27(Kip1) and rescued expression levels and localization of adherence junction molecules to cell-cell contacts. These observations suggest involvement of JNKs in TM4SF5-mediated p27(Kip1) Ser10 phosphorylation and localization during epithelial-mesenchymal transition.

Canine behavioral problems and their effect on relinquishment of the Jindo dog.

The purpose of this study was to evaluate behavior problems of the Jindo dog, the native dog of Korea, based on an owner's survey and their effect on pet relinquishment. To live a better life with their own pet and prevent relinquishment, it is important to understand the innate behavior characteristics of dog breed and the potential causes of relinquishment. Information concerning various factors and demonstration of the five most common behavior problems was collected via 189 completed questionnaires. No factors significantly affected the demonstration of behavior problem. A total 151 of 189 dogs had behavior problems (79.9%) and 38 dogs did not have behavior problems (20.1%). Among 151 dogs, 139 dogs showed single behavior problem (92.1%). They were 'excessive excitability' (46.8%), 'excessive vocalization' (30.2%), 'inappropriate elimination' (17.3%), 'destructive behavior' (4.3%), and 'aggressive behavior' (1.4%), respectively. In addition, 12 dogs showed two concurrent behavior problems (7.9%) According to the results, the relinquishment of Jindo dogs was not significantly associated with canine behavior problems, which is the single greatest risk factor of relinquishment in general. The possible reasons for potential behavior problems include improper raising, lack of socialization, and insufficient dog training classes, therefore canine behavior would be improved by owner education.

Response to a combined treatment program with clomipramine, behavioral, and environmental management of compulsive tail chasing in a German Shepherd.

A 15-month-old female German Shepherd dog showing compulsive tail chasing was treated with a treatment protocol of pharmacological therapy (using clomipramine) in conjunction with behavioral and environmental management. The responses to the treatment protocol were assessed once a week for 7 weeks in respects to behavioral conditions and the frequency of bouts and its duration. The behavior history including mother, father, and her littermates were also evaluated for the apparent diagnosis. The compulsive tail chasing of the patient dog was improved on the aspect of behavioral condition and gradually decreased in the frequency and duration of bouts. However, in the mid of treatment, the animal showed an aggressive behavior as a concurrent sign associated with compulsive tail chasing.