Recent studies have suggested that loss of RUNX3 expression is involved with gastric tumor metastasis. However, the precise mechanism of RUNX3-mediated suppression of tumor metastasis remains elusive. We aimed to clarify the effect of RUNX3 on tumor metastasis in gastric cancer cell lines and tumors. Immunohistochemistry revealed that RUNX3 was significantly decreased in metastatic gastric cancer. Gelatin zymography and Western blot showed that instead of regulating matrix metalloproteinase 9 (MMP9) expression, RUNX3 expression inhibited MMP9 enzyme activity, and this was consistent with the upregulation of tissue inhibitor of metalloproteinases 1 (TIMP1) by RUNX3. TIMP1 siRNA treatment impaired RUNX3-mediated suppression of gastric cancer cell invasion. Reporter assays demonstrated regulation of TIMP-1 by RUNX3. Two RUNX3 binding sites were identified in the TIMP-1 promoter and direct interaction of RUNX3 with the TIMP-1 promoter was confirmed in vitro and in vivo. These findings provide evidence for RUNX3-mediated suppression of gastric cancer invasion and metastasis and define a novel molecular mechanism that for the metastasis-inhibiting activity of RUNX3. These data may be applied in the development of RUNX3 for gastric cancer metastasis diagnostics and therapeutics.Gastic cancer is second only to lung cancer as a cause of cancer-related deaths worldwide, and poor prognosis and high mortality continue to be problems with this disease. 1-3 Approximately 20% of patients survive to 5 years, 4 and metastasis is a major cause of mortality in gastric cancer patients. Clearly, improvements in treatment depend on understanding the molecular mechanisms of gastric cancer metastasis. In general, cancer metastasis consists of a series of sequential, interrelated events involving growth factors, cell-adhesion molecules, matrix-degradation enzymes and motility factors. These molecules induce not only cell growth but also the extracellular matrix (ECM) degradation and angiogenesis that are required for tumor invasion and proliferation. 5 Previous research on the molecular pathology of gastric cancer metastasis identified a variety of key oncogenes and tumor suppressor genes in this process, such as c-erbB2, c-met, p27 and pRb. [6][7][8][9][10][11] Recently, deficiency in the Runt-related gene RUNX, which is a candidate tumor suppressor, was found to be causally related with gastric cancer. 12 The RUNX family members, RUNX1, RUNX2 and RUNX3, encode DNA-binding a subunits that bind a common b subunit, CBFb, to generate heterodimeric transcription regulators. 13 All three RUNX family members play important roles in normal developmental processes and carcinogenesis. 14-18 Recent studies showed that RUNX3 expression levels are downregulated in gastric cancer, either from hemizygous deletion or from promoter methylation of the RUNX3 gene. Furthermore, a decrease in RUNX3 protein expression is significantly associated with decreased survival of gastric cancer patients. Li et al. 12 showed that RUNX3 expression was decreased in ...