Inhibition of mTORC1 signaling reduces tumor growth but does not prevent cancer progression in a mouse model of thyroid cancer

Abstract

Selective drugs targeting dysregulated oncogenic pathways are promising cancer therapies. Because the mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in human follicular thyroid cancer (FTC), we hypothesized that its inhibition could block cancer development and progression. We, therefore, analyzed the effect of a treatment with a specific mTORC1 inhibitor (RAD001) in a faithful mouse model of FTC with constitutive mTORC1 activation (TRbeta(PV/PV)Pten(+/-) mice). The treatment did not prevent capsular and vascular invasion of the thyroid and the occurrence of lung metastasis. However, it substantially decelerated thyroid tumor growth, thereby prolonging TRbeta(PV/PV)Pten(+/-) mouse life span. RAD001 efficiently inhibited mTORC1 activity, as shown by the reduced phosphorylation of its downstream targets involved in the activity of the translation machinery, such as ribosomal S6 kinase (p70(S6K)), eukaryotic translation initiation factor 4E binding protein (4E-BP1) and the eukaryotic translation initiation factors eIF-4B and eIF-4G. Whereas mTORC1 signaling inhibition did not alter cell apoptosis, it induced a significant decrease in cell proliferation that was associated with the reduced abundance and altered activity of key regulators of cell cycle progression. Altogether, our data indicate that mTORC1 signaling plays a major role in the integration of the mitogenic signal in FTC. Therefore, our preclinical study with a relevant mouse model of FTC demonstrates for the first time that RAD001 efficaciously stabilizes cancer growth although it does not prevent its fatal outcome. In conclusion, our work underscores that in the treatment of FTC patients, RAD001 can only be used in combination with drugs and therapies inducing tumor shrinkage and blocking metastasis.

Figures

Fig. 1.

mTORC1 signaling inhibition by RAD001 augments life span by decreasing thyroid tumor growth. (A) Kaplan–Meier survival curves for TRβPV/PVPten+/− mice treated or not with RAD001. TRβPV/PVPten+/− mice received the placebo or RAD001 (10 mg/kg body wt) twice a week by oral gavage from 6 weeks of age until they had to be euthanized because of sickness. (B) Thyroid glands of TRβPV/PVPten+/− mice, treated or not with RAD001, were dissected and weighed. The data are presented as ratios of thyroid weight to body weight. The difference in the thyroid weight between TRβPV/PVPten+/− mice treated or not with RAD001 is significant (P < 0.001), as determined by Student's t-test analysis. (C) Body weights of TRβPV/PVPten+/− mice receiving placebo or RAD001. Student's t-test analysis did not show any significant difference (P = 0.096; NS, not significant).

Fig. 2.

Inhibition of mTORC1 signaling reduces cell proliferation without affecting cell apoptosis in FTC. (A) Representative microphotographs of Ki-67 immunohistochemistry on sections of placebo- and RAD001-treated TRβPV/PVPten+/− thyroids counterstained by hematoxylin; bar, 50 μm. (B) Thyroid cell proliferative index, determined by Ki-67 immunohistochemistry in the two groups, shows that there is a significant reduction in the percentage of proliferating cells in the thyroids of RAD001-treated TRβPV/PVPten+/− mice (n = 4 mice) as compared with placebo-treated TRβPV/PVPten+/− mice (n = 5 mice). (C) Representative microphotographs showing no increase in apoptosis in RAD001-treated animals after prolonged treatment, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Involuting wild-type female mammary gland, used as a positive control for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, shows apoptotic epithelial cells (depicted by arrows); bar, 50 μm.

Fig. 3.

mTORC1 inhibition does not block capsular and vascular invasion of the thyroid and metastasis in TRβPV/PVPten+/− mice. (A) Hematoxylin and eosin staining of thyroids (top and middle rows) and lungs (lower rows) from TRβPV/PVPten+/− mice receiving the placebo or RAD001. Hyperplastic thyroids show capsular invasion in the surrounding tissue in placebo-treated TRβPV/PVPten+/− mice (top row, in the muscle, shown as m) and in RAD001-treated TRβPV/PVPten+/− mice (top row, in the fat tissue, shown as f). Both groups displayed vascular invasion as shown by the thyroid cells in vessels (pointed by the arrows in middle rows). Lung metastasis was observed in both placebo- and RAD001-treated TRβPV/PVPten+/− mice (depicted by arrows in lower rows). (B) Sections of thyroids and lungs from placebo- and RAD001-treated TRβPV/PVPten+/− mice were stained with hematoxylin and eosin and analyzed for pathological progression of thyroid cancer, i.e. advanced hyperplasia, capsular invasion, vascular invasion of the thyroid and metastasis spread to the lung. The data are expressed as the percentage of occurrence of total mutant mice examined; NS, not significant (P > 0.05).

Fig. 4.

RAD001 efficiently suppresses mTORC1 signaling and reduces the abundance of Cyclin D3 and the phosphorylation of pRb. (A) The mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. By interacting with mTORC1- or mTORC2-specific proteins, mTOR acquires different substrate specificities and cell function. mTORC2 directly phosphorylates and activates Akt. In contrast, mTORC1 is phosphorylated and activated by the PI3K/Akt pathway, and in turn, it phosphorylates a number of target proteins involved in messenger RNA translation, such as the 4E-BP1, the eukaryotic translation initiation factor (eIF)-4G (eIF-4G) and p70S6K. Upon mTORC1 activation, hyperphosphorylated 4E-BP1 dissociates from eIF-4E, resulting in the recruitment of the scaffold protein eIF-4G to the 5′ end of the messenger RNA and thereby allowing translation initiation to proceed. mTORC1 also phosphorylates eIF-4G, and this may increase eIF-4G activity. The activation of p70S6K leads to increased phosphorylation of the ribosomal protein S6 (S6) and to the hyperphosphorylation of eIF4-B, which then promotes the ‘unwinding’ of the RNA secondary structures. Thus, mTORC1 is a master regulator of components of the translation machinery, thereby controlling protein synthesis. (B) Total protein extracts were prepared from thyroids of wild-type mice (lanes 1 and 2) and thyroid tumors of TRβPV/PVPten+/− mice (lanes 3–6) treated or not with RAD001, as described in Materials and Methods. Western blot analysis of phosphorylated p70S6K, total p70S6K, phosphorylated S6, total S6, phosphorylated 4E-BP1, total 4E-BP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The ratios of phosphorylated protein to total protein levels, after quantification of the band intensities for each sample, are indicated. (C) Total protein extracts were prepared from thyroid tumors of TRβPV/PVPten+/− mice receiving the placebo (lanes 1–3) or RAD001 (lanes 3–6), as described in Materials and Methods. Western blot analysis of phosphorylated eIF-4B, phosphorylated eIF-4G, total eIF-4G and GAPDH as a loading control. The ratios of phosphorylated eIF-4B to GAPDH and those of phosphorylated eIF-4G to total eIF-4G protein levels, after quantification of the band intensities for each sample, are indicated. (D) Total protein extracts were prepared from thyroid tumors of TRβPV/PVPten+/− mice receiving the placebo (lanes 1–3) or RAD001 (lanes 3–6), as described in Materials and Methods. Western blot analysis of Cyclin D1, Cyclin D3, phosphorylated pRb, total pRb and GAPDH as a loading control; ns, non-specific band. (E) Western blot analysis of Bax, MMP-2 and GAPDH as a loading control.

Fig. 5.

mTORC1 signaling inhibition by RAD001 does not alter PI3K/Akt and mitogen-activated protein kinase (ERK1/2) pathways in the thyroid tumors of TRβPV/PVPten+/− mice. Total protein extracts were prepared from thyroid tumors of TRβPV/PVPten+/− mice receiving the placebo (lanes 1–3) or RAD001 (lanes 3–6), as described in Materials and Methods. (A) Western blot analysis of phosphorylated Akt, total Akt, phosphorylated mTOR, total mTOR, phosphorylated GSK3β, total GSK3β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The ratios of phosphorylated protein to total protein levels, after quantification of the band intensities for each sample, are indicated. (B) Western blot analysis of phosphorylated ERK1/2, total ERK1/2 and GAPDH as a loading control.

Fig. 6.

Proposed model for the effect of the inhibition of mTORC1 signaling by RAD001 in the thyroids of TRβPV/PVPten+/− mice. RAD001 significantly inhibits the activity of the mTORC1 downstream targets belonging to the translation initiation machinery. Whereas the treatment did not inhibit cell migration to prevent cancer progression or exert noticeable proapoptotic actions, it consistently reduced cell proliferation, thereby reducing thyroid cancer growth to augment mouse life span.