Destabilizing RET in targeted treatment of thyroid cancers

Abstract

Metastatic differentiated thyroid cancers (DTC) are resistant to traditional chemotherapy. Kinase inhibitors have shown promise in patients with progressive DTC, but dose-limiting toxicity is commonplace. HSP90 regulates protein degradation of several growth-mediating kinases such as RET, and we hypothesized that HSP90 inhibitor (AUY922) could inhibit RET-mediated medullary thyroid cancer (MTC) as well as papillary thyroid cancer (PTC) cell growth and also radioactive iodine uptake by PTC cells. Studies utilized MTC cell lines TT (C634W) and MZ-CRC-1 (M918T) and the PTC cell line TPC-1 (RET/PTC1). Cell viability was assessed with MTS assays and apoptosis by flow cytometry. Signaling target expression was determined by western blot and radioiodine uptake measured with a gamma counter. Prolonged treatment of both MTC cell lines with AUY922 simultaneously inhibited both MAPK and mTOR pathways and significantly induced apoptosis (58.7 and 78.7% reduction in MZ-CRC-1 and TT live cells respectively, following 1 μM AUY922; P<0.02). Similarly in the PTC cell line, growth and signaling targets were inhibited, and also a 2.84-fold increase in radioiodine uptake was observed following AUY922 administration (P=0.015). AUY922 demonstrates in vitro activity against MTC and PTC cell lines. We observed a potent dose-dependent increase in apoptosis in MTC cell lines following drug administration confirming its anti-tumorigenic effects. Western blots confirm inhibition of pro-survival proteins including AKT suggesting this as the mechanism of cell death. In a functional study, we observed an increase in radioiodine uptake in the PTC cell line following AUY922 treatment. We believe HSP90 inhibition could be a viable alternative for treatment of RET-driven chemo-resistant thyroid cancers.

Keywords: AUY922; HSP90; MAPK; RET; mTOR; thyroid cancer.

Figure 1

Cells were treated for 48 and 72 h with increasing concentrations of AUY922. After 2 h of MTS incubation, absorbance was read at 490 nm. Representative experiment performed in triplicate at 72 h. (A) Percent growth inhibition was calculated. Inhibition was statistically significant in both cell lines in concentrations of 25 nM and above (P<0.002) (B) IC50 values were calculated on GraphPad Prism version 6 and are expressed in nM.

Figure 2

Cell cycle analysis of MTC cell lines after AUY922 treatment. (A) TT cells untreated. (B) TT cells treated with 250 nM AUY922. Cells demonstrate no change in cell cycle components following treatment of compound. (C) MZ-CRC-1 cells untreated. (D) MZ-CRC-1 cells treated with 250 nM AUY922 for 72 h. There is an increase in G-1 proportion following treatment. Modeling of cell cycle distribution was performed using ModFit LT software. The percentage of cells in each cell-cycle stage are shown for (E) TT and (F) MZ-CRC-1 cells. There was only a significant difference (*P<0.05) in G0/G1 proportion in MZ-CRC-1 cells following treatment.

Figure 3

Protein expression in MTC cell lines following AUY922 treatment. Cells were treated with increasing concentrations of AUY922 for 24 h. Cells were lysed and prepared for western blotting. Membranes were probed by antibodies, for RET and downstream targets of mTOR and MAPK pathway. Treatment with AUY922 inhibited phosphorylation of RET, ERK, RS6, and AKT in a dose-responsive manner.

Figure 4

DilC(5) vs PI flow cytometry analysis on MTC cell lines. (A and B) DilC(5) vs PI flow cytometry analysis on MTC cell lines A:TT and B: MZ-CRC-1 show increased apoptosis following AUY922 treatment. Cells were treated with increasing concentrations of AUY922 for 6 days. Cells were stained with DilC(5) and PI and analyzed on a FACS Calibur flow cytometer. Analysis was performed by Cell Quest Pro. (A and B) are representative examples of individual experiments which were completed three times. (C and D) Combined flow cytometry results for dose response of AUY922. Each individually treated cohort was normalized to vehicle treated cells on that experiment (*P<0.05, **P<0.01).

Figure 5

Protein expression and cell viability in the TPC-1 cell line following AUY922 treatment. (A) Cells were treated with increasing concentrations of AUY922 for 24 h. After 2 h MTS incubation, absorbance was analyzed at 490 nm. Graphs of log (inhibitor) vs response and analysis of nonlinear fit are shown at 24 h. IC50 values were calculated on GraphPad Prism version 6. (B) Cells were treated with increasing concentrations of AUY922 for 24 h. Cells were lysed and prepared for western blotting. Membranes were probed for antibodies to RET and downstream targets of the mTOR and MAPK pathways. Treatment with AUY922 inhibited phosphorylation of RET, ERK, RS6, and AKT in a dose-responsive manner. Furthermore, increasing doses of the AUY922 negatively impacted upon total RET and ERK protein expression.

Figure 6

Radioiodine uptake is increased in TPC-1 cell lines following AUY922 administration. Cells were plated to 70% confluence and then treated with increasing concentrations of AUY922 in triplicate. The following day cells were incubated with Na¹²⁵I or Na¹²⁵I and KClO₄ at 37 °C for 1 h. Cells were washed with 1× Hanks Buffer and solubilized with NaOH overnight at 4 °C. The following day cells were counted on gamma counter. There is significant increase in uptake at 1 μM AUY922 in both conditions (*P<0.05).