Inhibition of thyroid cancer proliferation in vitro by a novel small molecule HSP90 inhibitor.

C239

Introduction: Thyroid cancer represents 1% of all malignancies worldwide with papillary cancers representing the majority of cases having a good prognosis. Poorly differentiated and metastatic thyroid cancer, however carry a poor survival due to lack of effective systemic therapies. Currently several agents that target the MAP kinase pathway or inhibit heat shock protein 90 (HSP90I) are undergoing human clinical trials. These agents have thusfar shown limited efficacy and moderate systemic toxicity profiles. The search for novel small molecule inhibitors with good efficacy and low toxicity is critical to advancing treatment of these cancers. The purpose of this study is to determine the antiproliferative properties and mechanism of action of a novel carboxy-terminal inhibitor of HSP90 in thyroid cancer cell-lines in vitro.
 Methods: Human thyroid cancer cell lines for papillary cancer (DRO), follicular cancer (FTC-133) and anaplastic cancer (NPA) were cultured in RPMI medium and exposed to varying concentrations of KU-135, a novel C-terminal inhibitor of HSP90, with controls (no inhibitor) for 24 to 72 hours. Cellular protein was analyzed by immunoprecipitated Western blot analysis probing with monoclonal antibodies to B-RAF and N-RAS. Cell proliferation was determined by MTS assay at 72 hours exposure and apoptosis induction was evaluated by annexinV-PI co-staining with flow cytometry with cells incubated with drug for 18, 24, 48 and 72 hours.
 Results: Cell proliferation analysis by MTS assay at 72 hours exposure to KU-135 demonstrates IC50 levels of 900 nM for DRO, 2 µM for FTC and 3 µM for NPA. Western blot analysis of DRO and NPA cells incubated with varying concentrations (500nM - 50 µM) of KU-135 demonstrate modulation of the MAP kinase pathway in a dose-dependant manner with initial effects occurring at 1 micromolar concentration of drug. Annexin V- PI costaining with flow cytometry analysis of all three cell lines demonstrate that over 30% of cell death observed is likely due to apoptosis. This occurred in a dose-dependant manner with gating of early apoptotic cells seen within 24 hours exposure to KU-135.
 
 Conclusions: KU-135 is a novel C-terminal HSP90 inhibitor which demonstrates antiproliferative effects in both differentiated and anaplastic thyroid cancer cell lines in a dose dependant manner with IC50 levels close to 1 µM. This inhibitory effect is almost 10-fold greater than other HSP90 inhibitors currently being used in clinical trials. Initial analysis of the mechanism of action of KU-135 in thyroid cancer suggests a combination of MAP kinase pathway modulation along with induction of apoptosis, although other client proteins and pathways may also be involved. While this study shows promising in vitro data for KU-135, the role of this agent as a potential novel targeted therapy in thyroid cancer will require further analysis of toxicity and efficacy in vivo.