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

Abstract

Purpose/Objective(s)Heat shock protein 90 (HSP90) is a molecular chaperone that plays a key role in the folding, stability and function of many oncogenic signaling proteins. HSP90 expression is elevated in a wide spectrum of malignancies including mammary carcinoma cells and its activity appears to be required in order to permit accumulation of over-expressed and mutated oncogenes. Preclinical anti-tumor activity of HSP90 inhibitors have been reported in several human cancer cell lines including triple positive and triple negative breast cancers. There is however, very limited data on the combination of radiation with HSP90 inhibitors in the treatment of breast cancers, and no data to date in inflammatory breast cancer. Since radioresistance is associated with increased expression of many of the client proteins of HSP90, we hypothesized that treatment with the novel HSP90 inhibitor, ganetespib, prior to irradiation would lead to radiosensitization in breast cancer cell lines as compared to either treatment alone.Materials/MethodsExperiments were conducted using the inflammatory breast cancer cell line SUM-149, and the triple negative cell line MDA-MB-231. To assess the effect of the drug alone on the proliferation of the two breast cancer cell lines, XTT assays were performed. Cells were subsequently treated with drug, ionizing radiation, or a combination of the two. Treated cells were then analyzed for colony-forming ability, cell-cycle distribution and expression of several marker proteins (HSP90, HSP70, AKT, p-AKT, pRb).ResultsWe observed a dependent response to ganetespib in the XTT assay for the SUM-149 cell line. At 150 nM, the viability was reduced to 41.5% indicating that this dose is near the IC50. Triple negative MDA-MB-231 cells showed no statistical difference from the control at lower concentrations (50, 100 nM) of drug. However, at 150 nM, the drug had a significant effect on the viability of the cells, reducing it to 61.3% of control (P < 0.0001). At 200 and 250 nM, the viability was reduced to 57.1% and 43.1%, respectively (P < 0.0001). Clonogenic assays revealed a significant radiosensitization effect of the drug in the SUM-149 cell line. Cell cycle analysis after 24 hr treatment with ganetespib resulted in a significant increase in the portion of SUM-149 cells in G0-G1 arrest. Western blot results demonstrated a reduction in p-AKT protein levels after treatment with ganetespib with no effect on HSP90 expression.ConclusionThese results indicate that ganetespib is a potent radiosensitizer in vitro. Its mechanism is multifactorial whereby it arrests the cell cycle in more radiosensitive stages, and also leads to the degradation of cell of proteins involved in the radioresistant phenotype. These effects overall lead to improved cell killing when combined with radiation. Purpose/Objective(s)Heat shock protein 90 (HSP90) is a molecular chaperone that plays a key role in the folding, stability and function of many oncogenic signaling proteins. HSP90 expression is elevated in a wide spectrum of malignancies including mammary carcinoma cells and its activity appears to be required in order to permit accumulation of over-expressed and mutated oncogenes. Preclinical anti-tumor activity of HSP90 inhibitors have been reported in several human cancer cell lines including triple positive and triple negative breast cancers. There is however, very limited data on the combination of radiation with HSP90 inhibitors in the treatment of breast cancers, and no data to date in inflammatory breast cancer. Since radioresistance is associated with increased expression of many of the client proteins of HSP90, we hypothesized that treatment with the novel HSP90 inhibitor, ganetespib, prior to irradiation would lead to radiosensitization in breast cancer cell lines as compared to either treatment alone. Heat shock protein 90 (HSP90) is a molecular chaperone that plays a key role in the folding, stability and function of many oncogenic signaling proteins. HSP90 expression is elevated in a wide spectrum of malignancies including mammary carcinoma cells and its activity appears to be required in order to permit accumulation of over-expressed and mutated oncogenes. Preclinical anti-tumor activity of HSP90 inhibitors have been reported in several human cancer cell lines including triple positive and triple negative breast cancers. There is however, very limited data on the combination of radiation with HSP90 inhibitors in the treatment of breast cancers, and no data to date in inflammatory breast cancer. Since radioresistance is associated with increased expression of many of the client proteins of HSP90, we hypothesized that treatment with the novel HSP90 inhibitor, ganetespib, prior to irradiation would lead to radiosensitization in breast cancer cell lines as compared to either treatment alone. Materials/MethodsExperiments were conducted using the inflammatory breast cancer cell line SUM-149, and the triple negative cell line MDA-MB-231. To assess the effect of the drug alone on the proliferation of the two breast cancer cell lines, XTT assays were performed. Cells were subsequently treated with drug, ionizing radiation, or a combination of the two. Treated cells were then analyzed for colony-forming ability, cell-cycle distribution and expression of several marker proteins (HSP90, HSP70, AKT, p-AKT, pRb). Experiments were conducted using the inflammatory breast cancer cell line SUM-149, and the triple negative cell line MDA-MB-231. To assess the effect of the drug alone on the proliferation of the two breast cancer cell lines, XTT assays were performed. Cells were subsequently treated with drug, ionizing radiation, or a combination of the two. Treated cells were then analyzed for colony-forming ability, cell-cycle distribution and expression of several marker proteins (HSP90, HSP70, AKT, p-AKT, pRb). ResultsWe observed a dependent response to ganetespib in the XTT assay for the SUM-149 cell line. At 150 nM, the viability was reduced to 41.5% indicating that this dose is near the IC50. Triple negative MDA-MB-231 cells showed no statistical difference from the control at lower concentrations (50, 100 nM) of drug. However, at 150 nM, the drug had a significant effect on the viability of the cells, reducing it to 61.3% of control (P < 0.0001). At 200 and 250 nM, the viability was reduced to 57.1% and 43.1%, respectively (P < 0.0001). Clonogenic assays revealed a significant radiosensitization effect of the drug in the SUM-149 cell line. Cell cycle analysis after 24 hr treatment with ganetespib resulted in a significant increase in the portion of SUM-149 cells in G0-G1 arrest. Western blot results demonstrated a reduction in p-AKT protein levels after treatment with ganetespib with no effect on HSP90 expression. We observed a dependent response to ganetespib in the XTT assay for the SUM-149 cell line. At 150 nM, the viability was reduced to 41.5% indicating that this dose is near the IC50. Triple negative MDA-MB-231 cells showed no statistical difference from the control at lower concentrations (50, 100 nM) of drug. However, at 150 nM, the drug had a significant effect on the viability of the cells, reducing it to 61.3% of control (P < 0.0001). At 200 and 250 nM, the viability was reduced to 57.1% and 43.1%, respectively (P < 0.0001). Clonogenic assays revealed a significant radiosensitization effect of the drug in the SUM-149 cell line. Cell cycle analysis after 24 hr treatment with ganetespib resulted in a significant increase in the portion of SUM-149 cells in G0-G1 arrest. Western blot results demonstrated a reduction in p-AKT protein levels after treatment with ganetespib with no effect on HSP90 expression. ConclusionThese results indicate that ganetespib is a potent radiosensitizer in vitro. Its mechanism is multifactorial whereby it arrests the cell cycle in more radiosensitive stages, and also leads to the degradation of cell of proteins involved in the radioresistant phenotype. These effects overall lead to improved cell killing when combined with radiation. These results indicate that ganetespib is a potent radiosensitizer in vitro. Its mechanism is multifactorial whereby it arrests the cell cycle in more radiosensitive stages, and also leads to the degradation of cell of proteins involved in the radioresistant phenotype. These effects overall lead to improved cell killing when combined with radiation.