7114

TMSB4X

thymosin beta 4, X-linked;TMSB4X;thymosin beta 4, X-linked

prothymosin beta-4|t beta-4|thymosin beta-4|thymosin, beta 4, X chromosome

Xq21.3-q22

protein-coding

Intratumoral hypoxia has been correlated with distant metastatic potential. Two hypoxia inducible factors (HIFs), HIF-1alpha and HIF-2alpha, are induced by hypoxia, and high expression of these proteins has been correlated to angiogenesis and distant metastasis. Thymosin beta4 (Tbeta4) is frequently highly expressed in cancer, and this overexpression correlates with malignant progression. The objective of this study was to investigate the clinical correlation of HIF-alpha with Tbeta4 and the intracellular functional roles of Tbeta4 on HIF-alpha activation. We examined HIF-1alpha, HIF-2alpha and Tbeta4 expressions in clinical human breast carcinoma (n=70) by immunohistochemistry. We show that high expression of HIF-1alpha and HIF-2alpha strongly correlates with Tbeta4 expression (Pexpression of Tbeta4 correlates significantly with patients with lymph node metastasis (P<0.05) of human breast cancer. Additionally, we demonstrate that hypoxia up-regulates intracellular Tbeta4 protein, which then affects HIF-alpha activity, which is the key in regulating VEGF expression. We confirmed that hypoxia-induced intracellular Tbeta4 and HIF-alpha activities were reduced by interference of Tbeta4 expression using Tbeta4 shRNA lentivirus. Vascular epidermal growth factor (VEGF)-A, a well-recognized lymphangiogenic cytokine, was also down-regulated, but VEGF-C and VEGF-D expressions were not affected. These findings suggest that the overexpression of Tbeta4 is strongly associated with HIF-1alpha and HIF-2alpha expression and is also clinicopathologically involved with lymph node metastatic potential of breast cancer through the modulation of HIF-alphaactivation and induction of VEGF-A. Ultimately, these results highlight Tbeta4 as a potentially therapeutic target in malignant cancers.

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the synthesis of polyamines essential for cell growth and proliferation. Its overexpression induces the transformation of murine fibroblasts in both sense and antisense orientations, yielding highly invasive tumors in nude mice. These cell lines hence provide a good model to study cell invasion. Here, the gene expression profiles of these cells were compared with their normal counterpart by microarray analyses (Incyte Genomics, Palo Alto, CA, and Affymetrix, Santa Clara, CA). Up-regulation of the actin sequestering molecule thymosin beta4 was the most prominent change in both cell lines. Tetracycline-inducible expression of thymosin beta4 antisense RNA caused a partial reversal of the transformed phenotype. Further, reversal of transformation by dominant-negative mutant of c-Jun (TAM67) caused reduction in thymosin beta4 mRNA. Interestingly, a sponge toxin, latrunculin A, which inhibits the binding of thymosin beta4 to actin, was found to profoundly affect the morphology and proliferation of the AdoMetDC transformants and to block their invasion in three-dimensional Matrigel. Thus, thymosin beta4 is a determinant of AdoMetDC-induced transformed phenotype and invasiveness. Up-regulation of thymosin beta4 was also found in ras-transformed fibroblasts and metastatic human melanoma cells. These data encourage testing latrunculin A-like and other agents interfering with thymosin beta4 for treatment of thymosin beta4-overexpressing tumors with high invasive and metastatic potential.

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-1alpha 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-1alpha 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-1alpha stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.