Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

Abstract

Background: The Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets.

Methods: The expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy.

Results: The results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar.

Conclusions: We demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment.

Figure 1

Correlation of Aurora kinases expression with the TNM stage and RET status. (A) The Aurora kinase mRNAs level in MTC tissues was quantified as described in the Materials and Methods section. The statistical analysis of differences in the expression level of the three kinases in MTC tissues at different TNM stages was assessed by the analysis of variance (ANOVA) followed by the Tukey post ANOVA test. (B) Aurora kinases mRNA level in MTC tissues harboring the wild type (WT) or the mutated (Mut) RET protein. The bars in the graphs indicate the median values.

Figure 2

Time- and dose-dependent effects of the MK-0457 on TT cell proliferation. The TT cells were cultured in absence (DMSO) or in presence of 200 nM MK-0457 for different periods of time (A) or with different concentrations of MK-0457 (5 nM - 1000 nM) for 6 days (B). Data reported are representative of one out of three similar experiments. Statistical significance of data was assessed by the Student t-test. * p < 0.01.

Figure 3

Effects of the MK-0457 on TT cell ploidy. Cells were incubated for 6 days with 200 nM MK-0457 or the vehicle (DMSO). At the end of the incubation time cells were fixed and analyzed by FACS. See also table 2. For the immunofluorescence experiments (insert) TT cells were exposed or not for 6 days to 200 nM MK-0457, then fixed and stained with DAPI and β-tubulin. Scale bar, 20 μm.

Figure 4

Time-lapse analysis of control and MK-0457 treated TT cells. Pictures of cells cultured in the absence or in the presence of 200 nM MK-0457 were recorded every 5 min during 24 h using the MetaVue software. Data reported are representative of one out of three similar experiments. Numbers in the inserts represent the minutes. Scale bar, 10 μm.

Figure 5

Effects of the MK-0457 on Aurora kinases expression, subcellular localization, centrosome maturation and histone H3 phosphorylation in TT cells. (A) Western blot analysis of Aurora kinases protein levels in TT cells treated or not with MK-0457 (200 nM) for 48 h. For immunofluorescence experiments TT cells were treated or not for 6 h with MK-0457 (200 nM). Cells have been stained for Aurora-A and β-tubulin (A), Aurora-B and P-Histone H3 (B) or for Aurora-C and β-tubulin (C). Scale bar, 10 μm.

Figure 6

Effects of the MK-0457 on TT cell colony formation in soft agar. (A) TT cells were plated in soft agar onto 3.5 cm Petri dishes in the absence or in the presence of MK-0457 (200 nM). Treated and non-treated plates were photographed after three weeks of incubation. The colony size was determined using the MetaVue software and those larger than 50 μm in diameter were scored. Photographs reported in the figure are representative of one out of three similar experiments each performed in triplicate. Scale bar, 100 μm. (B) Effects of MK-0457 on the number and size of TT colonies in soft agar. Data reported represent the mean ± SEM of three independent experiments. *p < 0.001