MSX2 inhibits the growth and migration of osteosarcoma cells by repressing SOX2.

SRY (sex determining region Y)-box 2 (SOX2) plays a key role in the maintenance of stemness and resistance to drugs, whereas tumor necrosis factor (TNF)-α is essential for maintaining cancer cell proliferation and metastasis. Accumulation of muscle segment homeobox 2 (MSX2) leads to downregulation of SOX2 expression. Here, we explored the MSX2-SOX2-TNF-α signaling axis and its function in the tumor phenotypes of osteosarcoma cells. Colony formation assay, cell counting kit (CCK)-8 assay, and Flow cytometry were used to examine cell growth, viability, and death, respectively. Wound healing and Transwell invasive assay were employed to examine cell migratory and invasive activities, respectively. Western blotting and RT-qPCR were used to determine the protein and mRNA expressions of MSX2, SOX2, TNF-α, Bax, and matrix metalloproteinase-2 (MMP-2). Osteosarcoma clinical samples and cells showed lower levels of MSX2 than normal healthy control samples. Overexpression of MSX2 led to a reduced activity of SOX2 and TNF-α, whereas MSX2 depletion did not contribute to upregulated SOX2 levels. A gain-of-function experiment showed that osteosarcoma cell viability and growth were reduced, cell death was increased, and migration and invasion were inhibited in the MSX2 overexpression group compared with those in the non-transfected group. Furthermore, co-overexpression of MSX2 and SOX2 counteracted the inhibitory effects of MSX2 on the abovementioned tumor phenotypes of osteosarcoma cells. An in vivo tumor growth assay showed that MSX2 overexpression slowed the growth rate of osteosarcoma xenograft tumors. Thus, MSX2 loss plays a crucial role in the osteosarcoma phenotype by elevating SOX2 and TNF-α levels. AJTR