TBX21 inhibits colorectal cancer metastasis through ARHGAP29/GSK3β inhibitory signaling- and MYCT1/ZO-1 signaling-dependent manner

T-box transcription factor 21 (TBX21) plays a vital role in regulating immune responses, systemic diseases, and tumor progression. However, the role of TBX21 in colorectal cancer (CRC) metastasis remains unclear. In this study, we observed that TBX21 expression was marked decreased in CRC tissues compared to normal tissues and was negatively correlated with TNM stages. Interestingly, CRC and normal cell lines lacked TBX21 expression. Ectopic expression of TBX21 inhibited CRC cell migration <i>in vitro</i> and metastasis <i>in vivo</i>. Furthermore, a human phospho-kinase array analysis indicated that TBX21 expression reduced phosphorylation of glycogen synthase kinase 3 beta (GSK3β). RNA sequencing identified Rho GTPase activating protein 29 (ARHGAP29) and MYC target 1 (MYCT1) as potential TBX21-related target genes. TBX21 directly binds to the ARHGAP29 promoter to upregulate ARHGAP29, which, in turn, inhibits GSK3β phosphorylation. Concurrently, TBX21 promotes MYCT1 expression, leading to interaction with ZO-1 to regulate the cytoskeleton. Together, the ARHGAP29/GSK3β and MYCT1/ZO-1 pathways suppress CRC cell metastasis. Conversely, knockdown of TBX21, ARHGAP29, or MYCT1, or LiCl application, which enhances GSK3β phosphorylation, counteracted TBX21-mediated inhibition of CRC cell migration <i>in vitro</i> and <i>in vivo</i>. These findings suggest TBX21 suppresses CRC metastasis, identifying it as a potential target for metastatic cancer treatment.