Drug-induced inhibition of HMGA and EZH2 activity as a possible therapy for anaplastic thyroid carcinoma

Abstract

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal neoplasms in humans, and just limited progresses have been made to extend patient survival and decrease ATC-associated mortality. Thus, the identification of novel therapeutic strategies for treating ATC is needed. Recently, our group has identified two proteins with oncogenic activity, namely HMGA1 and EZH2, with pivotal roles in ATC cancer progression. Therefore, we tested the ability of trabectedin, a HMGA1-targeting drug, and GSK126, an inhibitor of EZH2 enzymatic activity, to impair cell viability of four ATC-derived cell lines. In the present study, we first confirmed the overexpression of HMGA1 and EZH2 in all ATC-derived cell lines and tissues compared to the normal primary thyroid cells and tissues. Then, treatment of the ATC cell lines with trabectedin and GSK126 resulted in a drastic induction of apoptotic cell death, which increased when the ATC cell lines were treated with a combination of both drugs. Conversely, normal primary human thyroid cells did not show any significant reduction in their viability when exposed to the same drugs. Noteworthy, both drugs induced the deregulation of EZH2- and HMGA1-controlled genes. Altogether, these findings propose the combination of trabectedin and GSK126 as possible novel strategy for ATC therapy.

Keywords: EZH2; GSK126; HMGA; Trabectedin; anaplastic thyroid carcinoma.

Figure 1.

EZH2 and HMGA1 expression levels are upregulated in ATC-derived cell lines and tissues. (a) HMGA1 and EZH2 mRNA expression levels in a panel of ATC-derived cell lines (ACT1, FRO, 8505c and SW1736). The expression levels of normal human thyroid-derived primary cells were set equal to 1. Data are shown as mean ± SD (n = 3) (b) HMGA1 and EZH2 protein expression in the same ATC-derived cell lines and in normal human thyroid-derived primary cells. 50 μg of total cellular lysate was loaded per lane in reducing conditions. GAPDH was used as protein loading control. NT = normal thyroid tissue. (c) Twelve ATC tissues were analyzed for HMGA1 and EZH2 expression with respect to three normal thyroid tissues. The mean of normal samples was set equal to 1. Data are shown as mean ± SD (n = 3).

Figure 2.

ET-743 and GSK126 affect cell viability of ATC-derived cell lines but not that of primary thyroid cells. (a) Dose–response curves of ACT1, FRO, 8505c, SW1736 ATC cell lines and normal primary thyroid cells treated with increasing concentrations of ET-743 and GSK126 for 72 h (n = 3). The cells were exposed to increasing concentrations of the drugs and then tested for viability by MTT assay. (b) ET-743 and GSK126 IC50 values for ATC cell lines.

Figure 3.

ET-743 and GSK126 synergistically induce ATC cell death. (a) effects of combination treatment with ET-743 and GSK126 on ATC cell viability by the MTT assay 72 h after exposure. (c) Combenefit mapped surface output for the drug combinations involving ET-743 and GSK126 using Loewe synergy model. These drugs synergistically inhibit 8505c cell growth. Cells were treated with ET-743 and GSK126 in a 5 × 5 concentration grid for 48 h, cell viability was determined by MTT assay. Data are shown as mean ± SD (n = 3) *p < 0.0.5; **p < 0.01; ***p < 0.001.

Figure 4.

ET-743 and GSK126 treatment induces deregulation of HMGA1- and EZH2-target genes. (a) CCNB and BCL2 mRNA expression levels in 8505c cell treated or not with ET-743. (b) E-cadherin, CDKN1A and PAX8 mRNA expression levels in 8505c cell treated or not with GSK126. Data are shown as mean ± SD (n = 3) *p < 0.0.5; **p < 0.01; ***p < 0.001.

Figure 5.

Effects of ET-743 and GSK126 on cell cycle progression on ATC cell lines. cell-cycle profile after 48 h of treatment with ET-743 and GSK126 analyzed by PI incorporation and flow cytometry in the ACT1, FRO, 8505c and SW1736 cell lines. Data are shown as mean ± SD (n = 3) *p < 0.0.5; **p < 0.01; ***p < 0.001.

Figure 6.

Effects of ET-743 and GSK126 combination on apoptosis. (a) Annexin V FITC-A vs. PI plots from the gated cells shows the populations corresponding to viable and non-apoptotic (annexin V – PI–), early (annexin V + PI–), and late (annexin V + PI+) apoptotic cells in SW1736 cell line. (B) Quantification of ACT1, 8505c, FRO and SW1736 apoptotic cells after 72 h of treatment with ET-743 and GSK126 alone or combination of the two drugs. Data are shown as mean ± SD (n = 3) *p < 0.05; **p < 0.01; ***p < 0.001.