Tetrathiomolybdate (TM), a specific copper chelator, has been shown to be a potent antiangiogenic and antimetastatic compound possibly through suppression of the NFkappaB signaling cascade. To further delineate the molecular mechanism of the anticancer effect of TM, we investigated whether TM has antineoplastic activity in the setting of genetic NFkappaB inhibition. In this study, SUM149 inflammatory breast carcinoma cells were transfected with a dominant-negative IkappaBalpha (S32AS36A) expression vector. Similar to TM-treated SUM149 cells, SUM149-IkappaBalphaMut clones secreted lower amounts of proangiogenic mediators, vascular endothelial growth factor, interleukin-1alpha, and interleukin-8 and exhibited a less invasive and motile phenotype. The reduction in the angiogenic and metastatic potential of SUM149-IkappaBalphaMut clones was not further affected by TM in vitro. SUM149-IkappaBalphaMut xenografts grew substantially slower and had less lung metastasis than SUM149 and SUM149-empty vector xenografts. The growth and metastatic potential of SUM149 and SUM149-empty tumors was significantly inhibited with systemic TM treatment, whereas TM had no further antitumor effect on the SUM149-IkappaBalphaMut tumors. Additionally, nuclear proteins isolated from TM-treated SUM149 tumors had lower NFkappaB binding activity, while AP1 and SP1 binding activities were unchanged. Taken together, these results strongly support that suppression of NFkappaB is the major mechanism used by TM to inhibit angiogenesis and metastasis.