Evaluating the HSP90 Inhibitor Ganetespib as a Radiosensitizing Agent in Breast Cancer Models In Vitro

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• Experiments were conducted using the inflammatory breast cancer cell line SUM-149, and the triple negative cell line MDA-MB-231. The BT-474 (ER/PR positive, HER2 positive) and MCF-7 (ER/PR positive, HER2 negative) cell lines underwent XTT testing. • XTT Assays: • Cells were plated in 96 well plates, exposed to ganetespib in concentrations ranging from 50-250 nM for 24 hrs. Media was changed and cells were incubated for 72 hrs. XTT assays were then performed. • Clonogenic Assays: • Cells were plated on 6-well plates, ganetespib was added to the media at 10 or 50 nM and allowed to incubate 20 hours. Cells were then irradiated and incubated for 4 more hours. Media was changed, and 14 days later, plates were fixed, stained, and colonies counted. • Cell Cycle Analysis: • Cells were plated in flasks and treated with media or media + ganetespib at a concentration of 150 nM. The cells were incubated for 24 hours. They were then harvested, fixed in 70% ice cold ethanol, washed in 1xPBS, and re-suspended in 1 mL of PI solution containing DNAse-free RNAse. The cells were analyzed with a Beckman Coulter Flow Cytometer. • Western Blots: • Cells were treated with 100 nM ganetespib for 24 hours, harvested, then analyzed for expression of several marker proteins (HSP90, HSP70, AKT, p-AKT, pRb) Conclusions