The HDAC Inhibitor TSA Inhibits Cell Proliferation, Induces Apoptosis and Down-Regulates HER2 Protein and Gene Expression as a Single Agent and in Combination with Trastuzumab in Trastuzumab-Sensitive and -Resistant Breast Cancer Cell Lines.

The HER2 antibody trastuzumab is the first biological therapy that has proven the value of HER2 blockade in breast cancer. However, not all patients respond to trastuzumab and relapses occur after this treatment. Histone deacetylase inhibitors (HDACIs) are active in HER2-amplified breast cancer but the role of HDACIs in trastuzumab-resistant breast cancer has not been addressed yet. We evaluated the activity and mechanism of action of trichostatin (TSA), a potent pan-HDACI in HER2-amplified breast cancer cell lines exhibiting either de novo or acquired resistance or sensitivity to trastuzumab.Cell lines BT474, SkBr3, MDA-MB361, MDA-MB453, UACC812, UACC893 and MCF10A (ctrl.) were used for all experiments. TSA and trastuzumab were used for proliferation assays using WST1 reagent (Roche). Flow cytometry and Immunoblotting were done to detect apoptosis and phosphorylation of Akt and Erk1/2. Immunoprecipitation was performed to study HER2 interaction with Hsp90 during HDACI treatment. Immunofluorescence analysis was applied to visualize HER2 localization and membrane trafficking. Microarray analysis was performed to detect changes in expression of apoptosis, cell cycle and transcription factor genes in BT474 trastuzumab-sensitive and –resistant cells.TSA inhibited growth of all cell lines with similar IC50s. Combining trastuzumab with TSA did not show synergy but trastuzumab did not seem to disturb the growth-inhibitory effect of HDACIs.TSA as a single agent or combined with trastuzumab inhibited cell survival, proliferation and induced apoptosis. We observed a decrease of HER2 protein, Akt and Erk1/2 phosphorylation while activation of caspase-3 and PARP increased during TSA treatment in trastuzumab-sensitive and -resistant cell lines but not in a normal breast cell line. In addition, we showed a consistent transcriptional effect of TSA on HER2 mRNA, apoptosis and cell cycle genes in both sensitive and resistant lines using Illumina gene expression profiling. We found downregulation of transcription factors with predicted HER2 promoter binding motifs, suggesting a potential role of these genes in HER2 downregulation in addition to the reported effect on mRNA stability. Some of these TFs may be involved in chromatin regulation. Finally, using confocal microscopy, we observed HER2 localized at large, ubiquitin-negative vesicular structures at the plasma membrane that were strongly reduced during TSA treatment. This effect is not caused by disruption of trafficking pathways but rather by a downregulation of HER2 protein and gene expression.In conclusion, TSA exhibits growth-inhibitory and pro-apoptotic activity in both trastuzumab-sensitive and –resistant, HER2 overexpressing breast cancer cells but not in cells derived from normal breast epithelia. HDACIs could represent an alternative therapeutic strategy in trastuzumab-resistant breast cancer. It is likely that HDACIs modulate HER2 activity on multiple levels including HER2 protein, transcriptional control, and chromatin structure. A better understanding of these mechanisms is essential to determine the role of HDACIs in HER2 positive breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3133.