Thymosin beta-4 (Tb4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tb4 and it is associated with Tb4-mediated migration in gastric cancer cells. Various expression level of Tb4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tb4. Cell migration was higher in SNU668 with a higher expression level of Tb4 than that in SNU638 with a lower Tb4. Although the level of phosphorylated(p)-GSK-3a (inactive), b-catenin, E-cadherin and E-cadherin:b-catenin complex was relatively higher, p-GSK-3b (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3a/b inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3a increased SNU638 cell migration in accordance with the reduction of E-cadherin:b-catenin complex formation through a decrease in b-catenin and E-cadherin. Expression level of GSK-3a/b, b-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tb4-siRNA and by the treatment with acetylatedserine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tb4, respectively. E-cadherin expression in SNU638 cells was decreased by b-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3a, b-catenin and E-cadherin. Taken together, data indicated that the expression of GSK3a, b-catenin and E-cadherin could be negatively regulated by Tb4-induced ERK phosphorylation. It suggests that Tb4 could be a novel regulator to control Wnt signaling pathways.When cancer cells are away from the original place to the secondary location, cancer cell transition happens by the course of cell migration and adhesion in which a wide variety of proteins are involved and form a complex structure accompanying the reorganization of actin cytoskeleton. Thymosin beta-4 (Tb4) proteins regulate intracellular signal transduction and cytoskeleton structure.Tb4 initially isolated from the thymus in 1981 is a small and naturally occurring 43-amino acid peptide present in all cells except erythrocytes. Tb4 is one of b-thymosins that are the most abundant member of highly conserved polar 5-kDa peptides.

Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.

Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME).We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively.These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

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