Although enhanced thymosin β4 (TMSB4X/Tβ4) expression is associated with tumor progression and metastasis, its tumor-promoting functions remain largely unknown. Here, it is demonstrated that TGFβ facilitates Tβ4 expression and leads to the activation of myocardin-related transcription factors (MRTF), which are coactivators of serum response factor (SRF) and regulate the expression of genes critical for the epithelial-mesenchymal transition (EMT) and tumor metastasis. In murine mammary gland cells (NMuMG), Tβ4 upregulation is required for full induction of a MRTF-regulated EMT gene expression program after TGFβ stimulation. Tβ4 levels are transcriptionally regulated via the novel -acting element AGACAAAG, which interacts with Smad and T-cell factor/lymphoid enhancer factor (TCF/LEF) to synergistically activate the Tβ4 promoter downstream of TGFβ. Murine skin melanoma cells (B16F0 and B16F1) also show the expression regulation of Tβ4 by Smad and TCF/LEF. Tβ4-knockout B16F1 (Tβ4 KO) clones show significantly diminished expression level of tumor-associated genes, which is regulated by the TGFβ/MRTFs pathway. In multiple human cancers, Tβ4 levels correlate positively with TGFβ1 and the tumor-associated gene expression levels through processes that respectively depend on TGFβ receptor 1 (TGFBR1) and MRTF expression. Kaplan-Meier survival analyses demonstrate that high Tβ4 expression associates with poor prognosis in an SRF expression-dependent manner in several cancers. In mice, Tβ4 KO clones show significantly decreased experimental metastatic potential; furthermore, ectopic expression of constitutively active MRTF-A fully restores the diminished metastatic activity. In conclusion, the TGFβ/Tβ4/MRTF/SRF pathway is critical for metastasis and tumor progression. These findings define a molecular mechanism underlying a tumor-promoting function of thymosin β4 through activation of MRTF/SRF signaling. .

Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.

The epithelial-to-mesenchymal transition (EMT) plays a vital role in carcinogenesis, invasion, and metastasis of many epithelial tumors including oral squamous cell carcinoma (OSCC), a common malignancy of the head and neck. However, the functional role of the actin-sequestering protein thymosin β4 (Tβ4) in the EMT in OSCCs remains unclear. Thus, we investigated whether overexpression of Tβ4 could induce in vitro tumorigenesis such as cell proliferation and anchorage independency and an EMT-like phenotype in OSCCs. Also, we examined whether it affects invasiveness and cell motility-associated signaling molecules. Tβ4-overexpressing OSCCs, SCC-15_Tβ4 and SCC-25_Tβ4, enhanced cell proliferation and colony formation. In addition, we observed that Tβ4 overexpression induced an EMT-like phenotype, accompanied by a decrease in expression of the epithelial cell marker E-cadherin and an increase in expression of mesenchymal cell markers vimentin and N-cadherin. Also, the expression level of Twist1, an EMT-inducing transcription factor, was significantly enhanced in SCC-15_Tβ4 and SCC-25_Tβ4 cells. Tβ4 overexpression augmented in vitro invasion and MMP-2 activity and enhanced the phosphorylation of paxillin and cortactin and expression of LIMK1. Taken together, these results suggest that Tβ4 overexpression could be one of the causes of tumorigenesis and progression in OSCCs. Further investigation on the Tβ4 molecule would encourage the development of specific targets for cancer treatment.

Thymosin β(4) (Tβ(4)) overexpression increases cell migration and tumor metastasis. Hence, understanding the mechanism of cancer cell migration induced by Tβ(4) may provide means to inhibit their metastasis. We demonstrated higher Rac1 activities and expression levels of IQGAP1 and ILK in highly migrated Tβ(4)-overexpressing SW480 cells. In addition, IQGAP1 formed a complex with ILK and knockdown of Tβ(4) simultaneously reduced ILK and IQGAP1 protein levels as well as their migration ability. These findings suggest that Tβ(4) increases migration of colon cancer cells via activating Rac1 by elevating IQGAP1/ILK complexes and IHC results illustrated a similar mechanism occurring in vivo.