High levels of the cyclooxygenase-2 (COX-2) protein have been associated with invasion and metastasis of breast tumors. Both prostaglandin E(2) (PGE(2)) and interleukin-8 (IL-8) have been shown to mediate the invasive activity of COX-2 in breast cancer cells. Here we expand these studies to determine how COX-2 uses PGE(2) and IL-8 to induce breast cancer cell invasion. We demonstrated that PGE(2) and IL-8 decreased the expression of the tumor suppressor protein Programmed Cell Death 4 (PDCD4). We hypothesized that suppression of PDCD4 expression is vital to the invasive activity of PGE(2) and IL-8. In MCF-7 cells overexpressing PDCD4 (MCF-7/PDCD4), PGE(2) and IL-8 failed to induce invasion, in contrast to the parental MCF-7 cells, thus indicating that PDCD4 blocks breast cancer cell invasion. MCF-7/PDCD4 cells produced higher levels of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) than the parental cells. Silencing TIMP-2 mRNA in MCF-7/PDCD4 cells reversed the anti-invasive effects of PDCD4, allowing PGE(2) and IL-8 to induce the invasion of these cells. Here we report the novel findings that suppression of PDCD4 expression is vital for the invasive activity of COX-2 mediated by PGE(2) and IL-8, and that PDCD4 increases TIMP-2 expression to inhibit breast cancer cell invasion.