Everolimus is an active agent in medullary thyroid cancer: a clinical and in vitro study

Abstract

Everolimus, an mTOR inhibitor, which has been demonstrated to induce anti-tumour effects in different types of neuroendocrine tumours, has never been evaluated in patients with medullary thyroid cancer (MTC). The aim of this study was to evaluate the in vitro and in vivo effects of everolimus in combination with octreotide in MTC. Two patients with progressive metastatic MTC and high calcitonin levels were treated with everolimus 5-10 mg/day. Both patients were under treatment with octreotide LAR at the study entry. An in vitro study was also performed to assess everolimus effects on MTC cell lines (TT and MZ-CRC-1 cells). A tumour response was observed in both patients. Serum calcitonin decreased by 86% in patient 1 and by 42% in patient 2. In TT and MZ-CRC-1 cells, everolimus induced a significant dose-dependent inhibition in cell proliferation. This effect seems to be related to a cell cycle arrest in G(0) /G(1) phase in both cell lines and to the induction of cellular senescence in TT cells. Everolimus in combination with octreotide may be active as anti-tumour therapy in patients with progressive metastatic MTC, suggesting to further evaluate this agent in MTC patients in a large prospective study.

Fig 1

Calcitonin and CEA serum concentrations in Patient 1 (A, C) and Patient 2 (B, D) because the start of the treatment with octreotide LAR 30 mg a month and then during treatment with everolimus at different doses.

Fig 2

Colour Doppler ultrasonography imaging of a laterocervical lymph node metastasis before (A) and after 3 months of treatment with everolimus 10 mg a day (B).

Fig 3

Effects of 6 days of incubation with or without everolimus on cell proliferation (A, B) and CT secretion (C, D) in TT (A, C) and MZ-CRC-1 (B, D). Values are expressed as the percentage of control (untreated cells) and represent the mean ± S.E.M. of at least three independent experiments in six replicates. *P < 0.001 and **P < 0.05 versus control.

Fig 4

Cell cycle analysis of TT (A, B) and MZ-CRC-1 (C, D) cells after 6 days of incubation with everolimus, respectively at the concentrations of 5 and 75 nM. Data are expressed as mean ± S.E.M. of the percentage of cells in the G₀/G₁ (A, C) and S (B, D) phase of three independent performed experiments, as compared with untreated control cells. Control values have been set to 100%. *P = 0.05, **P < 0.05 and ***P < 0.001 versus untreated control.

Fig 5

Effect of everolimus on apoptosis in human MZ-CRC-1 cell line. MZ-CRC-1 cells were incubated with 75 nM of everolimus for 6 days. Subsequently, the proportions of cells in early (A) and late (B) apoptosis were analysed by flow cytometry. Values are demonstrated as average and S.E.M. of three independent performed experiments. During this experiment total cell lysates were analysed by Western blot to detect the cleavage of caspases 3, 8, 9 and PARP (C). Actin was used as control for equal lading.

Fig 6

Effect of everolimus on cellular senescence. TT (A) and MZ-CRC-1 (B) cells were treated with everolimus for 6 days at the concentrations of 5 and 75 nM, respectively, and then were stained for β-galactosidase. The results were reported as the percentage of β-galactosidase positive cells. The experiments were repeated at least three times. *P < 0.001.