Abstract Numerous studies indicate that deregulation of histone acetylation and deacetylation plays an important role in aberrant gene expression in human cancers. The Histone deacetylases (HDAC) have become attractive therapeutic targets, and many inhibitors of HDAC are actively under investigation as potential anti-cancer agents. SNDX-275 is an oral, class I HDAC inhibitor currently in multiple phase II clinical trials. We have recently reported that SNDX-275 selectively induces apoptosis of HER2-overexpressing breast cancer cells. Herceptin®, a humanized monoclonal antibody against HER2 receptor, has been successfully used in breast cancer patients with HER2-overexpressing tumors. In the current studies, we have investigated the potential synergism of SNDX-275 and Herceptin on inducing growth inhibition and/or apoptosis in HER2-overexpressing breast cancer cells, focusing on their combinational effects on the expression of both HER2 and HER3, and inactivation of downstream signaling pathways. The cell proliferation (MTS) assays showed that SNDX-275 significantly enhanced Herceptin-induced growth inhibition in HER2-overexpressing breast cancer cells as compared to the treatment with either agent alone. While SNDX-275 (0.5 µM) alone clearly decreased the levels of HER3 with little effect on HER2 levels, Herceptin (20 µg/ml) alone mainly lowered the expression levels of HER2 in the HER2-overexpressing SKBR3, BT474, and MDA-MB-453 cells. The combinations of SNDX-275 (0.5 µM) and Herceptin (20 g/ml) resulted in a dramatic reduction of both HER2 and HER3 levels, and subsequently led to inactivation of HER2 tyrosine kinase evidenced by western blot analysis showing reduced levels of both P-HER2 and P-HER3. On the downstream signaling pathways, neither the combination nor either agent alone had inhibitory effects on MAPK. In contrast, the combinations of SNDX-275 and Herceptin significantly decreased the levels of P-Akt in SKBR3, BT474, and MDA-MB-453 cells as compared to either agent alone. It appeared SNDX-275 induced expression of p21waf1, and Herceptin upregulated p27kip1, however, their combinations had no further inductions on either of these two CDK inhibitors. Moreover, apoptotic-ELISA and western blot analysis on PARP cleavage revealed that the combinations of SNDX-275 and Herceptin promoted more cells undergoing apoptosis as compared to SNDX-275 alone. Taken together, our results indicated that SNDX-275 and Herceptin synergistically induced growth inhibition and apoptosis in HER2-overexpressing breast cancer cells through combined downregulation of HER2/HER3 levels, inhibition of HER2 tyrosine kinase, and inactivation of the PI-3K/Akt signaling. These data suggest that further evaluation of SNDX-275 in HER2 positive breast cancer may be warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A187.