I. Lou1, X. Yu1, S. Jang1, A. Harrison1, H. Chen1 1University Of Wisconsin,Endocrine Surgery,Madison, WI, USA
Introduction:
Aberrant histone deacetylase (HDAC) activity has been shown to contribute to cancer development and HDAC inhibitors (HDACi) are currently under clinical investigation to treat a variety of malignancies. Despite the promising drug efficacy shown in preclinical studies, most HDACi, including suberoylanilide hydroxamic acid (SAHA), have failed to demonstrate significant survival benefit for metastatic thyroid cancers in clinical trials. In our current study, we aim to delineate the possible mechanism responsible for HDACi resistance in metastatic thyroid cancer cells through the development and characterization of a SAHA-resistant cell line.
Methods:
FTC236 cells, a human follicular thyroid cancer derived cell line metastatic to the lymph nodes, were chronically exposed to SAHA at increasing concentrations and maintained in 2µM SAHA containing medium (FTC236/SAHA-R). Parental cells exposed only to dimethyl sulfoxide (DMSO), the solvent for SAHA, were used as control (FTC236/DMSO). Cell proliferation was then measured by viable cell count. HDACi resistance was evaluated by treating the two cell lines with different increasing concentrations of SAHA. Protein lysate was collected from FTC236/SAHA-R and FTC236/DMSO. Western blotting was performed to assess the expression of Notch 1 protein as well as epithelial mesenchymal transition (EMT) markers, which have previously been shown to play a role in cancer chemoresistance.
Results:
Based on the viable cell counts, the FTC236/SAHA-R cell line consistently showed a slower growth rate than FTC236/DMSO (221% vs. 550% at 72 hours, p<0.05). FTC236/SAHA-R was found to be resistant not only at the established drug concentration of 2 µM but also at higher concentrations. After 48 hours of SAHA exposure, 58% and 35% of FTC236/SAHA-R remained viable at 2 μM and 4 μM concentrations, respectively. In comparison, only 35% and 23% of FTC236/DMSO survived in 2 μM and 4 μM SAHA, respectively. Analysis of the protein expression of these cell lines as well as the parental cell line revealed consistent silencing of Notch 1 signaling in the FTC236/SAHA-R cells. With respect to EMT markers, increased expression of Snail in FTC236/SAHA-R cells was observed compared with parental cell line and FTC236/DMSO while Slug remained unchanged.
Conclusion:
SAHA resistant thyroid cancer cells grow at a much slower rate. In addition, Notch 1 signaling is silenced and the expression of EMT marker Snail is increased in these resistant cells. This may shed light into possible mechanisms of HDAC inhibitor resistance and help with the development for new treatment targets.
