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Enhancing the anti-tumor activity of ErbB blockade with histone deacetylase (HDAC) inhibition.

3029 Background: Molecular inhibition of the ErbB signaling pathway is under active investigation as a promising anti-cancer treatment strategy. Pre-clinical studies suggest that enhanced anti-tumor activity may be achieved by maximizing ErbB signaling inhibition. Using high throughput screening with cDNA microarrays, we identified HDAC inhibitors as having strong potential to enhance the effects of anti-ErbB agents.


METHODS
Microarray: Total RNA was isolated from cells after 12 and 24 hours exposure to HDAC inhibitor SAHA (suberoylanilide hydroxamic acid). cDNA probes were prepared from experimental and appropriate reference RNA samples, labeled, and hybridized to 20K human cDNA. Data was validated using quantitative real time RT-PCR. Cell Proliferation: Exponentially growing cells were incubated in medium containing vehicle control, CI-1033 (pan-ErbB tyrosine kinase inhibitor), SAHA, or both drugs in combination for 72 hours. Cells were then stained with 0.5% crystal violet and cell viability was determined using relative absorbance. Apoptosis: Caspase activity was analyzed by fluorescence spectroscopy using a fluorescein labeled pan-caspase inhibitor.


RESULTS
Array studies using SAHA in prostate and breast cancer cell lines show decreased transcript expression (> 50%) of EGFR and ErbB2. Findings from the array data were validated using real time RT-PCR and western blot analysis. Enhanced proliferative inhibition was seen in prostate (DU145, PC3), breast (SKBr3, MCF7), and head and neck tumor cells (UM-SCC6) when SAHA was combined with CI-1033. Combining HDAC inhibition with EGFR blockade demonstrated strong abrogation of EGFR and AKT signaling following EGF stimulation, as well as a supra-additive increase in caspase activity (p<0.05).


CONCLUSIONS
These results suggest that used cooperatively, anti-ErbB agents and HDAC inhibitors may offer a unique and potentially potent strategy of dual targeted therapy. Based on emerging preclinical data, we are currently designing Phase I clinical trials which combine HDAC inhibitors with ErbB blockade. No significant financial relationships to disclose.