The NF1 tumor suppressor critically regulates TSC2 and mTOR

Abstract

Loss-of-function mutations in the NF1 tumor suppressor gene underlie the familial cancer syndrome neurofibromatosis type I (NF1). The NF1-encoded protein, neurofibromin, functions as a Ras-GTPase activating protein (RasGAP). Accordingly, deregulation of Ras is thought to contribute to NF1 development. However, the critical effector pathways involved in disease pathogenesis are still unknown. We show here that the mTOR pathway is tightly regulated by neurofibromin. mTOR is constitutively activated in both NF1-deficient primary cells and human tumors in the absence of growth factors. This aberrant activation depends on Ras and PI3 kinase, and is mediated by the phosphorylation and inactivation of the TSC2-encoded protein tuberin by AKT. Importantly, tumor cell lines derived from NF1 patients, and a genetically engineered cell system that requires Nf1-deficiency for transformation, are highly sensitive to the mTOR inhibitor rapamycin. Furthermore, while we show that the activation of endogenous Ras leads to constitutive mTOR signaling in this disease state, we also demonstrate that in normal cells Ras is differentially required for mTOR signaling in response to various growth factors. Thus, these findings identify the NF1 tumor suppressor as an indispensable regulator of TSC2 and mTOR. Furthermore, our results also demonstrate that Ras plays a critical role in the activation of mTOR in both normal and tumorigenic settings. Finally, these data suggest that rapamycin, or its derivatives, may represent a viable therapy for NF1.

Fig. 1.

The mTOR pathway is aberrantly regulated in Nf1^-/- MEFs. (A) Western blot analysis of serum-starved primary Nf1^+/+ and Nf1^-/- MEFs. Abbreviations are as follows: NF1, neurofibromin; pS6K, p70S6K phosphorylated at T389; pAKT, Akt phosphorylated at S473; RAPA, rapamycin. Cells were serum-starved and treated with rapamycin (20 nM) for 30 min where indicated. (B) Western blot analysis of primary Nf1^+/+ and Nf1^-/- MEFs deprived of amino acids (AA).

Fig. 2.

mTOR activation in normal and Nf1^-/- fibroblasts requires active Ras. (A) Ras GTP levels were quantitated by using a pull-down assay in cells expressing either the NF1-GRD or empty vector (Left). GTP-bound Ras, isolated from the pull-down assay (PD), and total Ras protein, present in the total cell extract (TCE), are shown. Western blot analysis of Nf1^+/+ and Nf1^-/- MEFs expressing the NF1-GRD or empty vector (Right). (B) Western analysis of serum-starved 3T3 fibroblasts expressing either the NF1-GRD or empty vector in response to 200 nM insulin (Top), 6 μM LPA (Middle), or 20 ng/ml PDGF (Bottom) for the indicated times.

Fig. 3.

Hyperactivation of the PI3 kinase pathway in Nf1^-/- primary MEFs results in tuberin inactivation. (A Left) Western blot analysis of serum-starved Nf1^+/+ and Nf1^-/- MEFs. Abbreviations are as follows: pS6K, p70S6K phosphorylated at T389; pAKT, AKT phosphorylated at S473; pTub-T1462, tuberin phosphorylated at T1462; pTub-S939, tuberin phosphorylated at S939. (A Right) Western blot analysis of tuberin phosphorylation in serum-starved Nf1^-/- MEFs after treatment with DMSO, rapamycin, or wortmannin. (B Left) HEK293 cells expressing either pLKO lentiviral vector or a short hairpin RNA (shRNA) against NF1 were transfected with an hemagglutinin-tagged p70S6K reporter. (B Right) HEK293 cells expressing shRNA against NF1 were transfected with the indicated tuberin or NF1-GRD constructs along with a hemagglutinin-tagged p70S6K reporter. Abbreviations are as follows: Tub-WT, wild-type tuberin; Tub-SATA, tuberin with alanine mutations at the AKT phosphorylation sites S939/T1462. Hemagglutinin immunoprecipitates (HA IP) and total cell extracts (TCE) were immunoblotted with the indicated antibodies.

Fig. 4.

NF1^-/- tumor cells exhibit mTOR hyperactivation and are highly sensitive to rapamycin. (A) Western blot analysis of two independently derived patient-matched NF1^+/- and NF1^-/- Schwann cells grown in 0.1% serum devoid of exogenous growth factors. (B) Western blot analysis of an NF1^-/- tumor cell line treated for 30 min with the following concentrations of inhibitors: DMSO, 0.1%; rapamycin, 20 nM; wortmannin, 200 nM. (C) NF1^-/- tumor cell lines were grown for 7 days in the presence of the indicated concentrations of rapamycin and counted in triplicate.

Fig. 5.

Rapamycin inhibits colony growth in soft agar of Nf1-deficient transformed cells. (A) Nf1^+/+ and Nf1^-/- MEFs retrovirally transduced with dominant negative p53 (pBabe-hygro-p53DD) and adenoviral E1A (pLPC-E1A12S) were selected and plated in soft agar. (B) Nf1^-/- MEFs (with the additional genetic alterations described in A) were grown in soft agar with the indicated concentration of rapamycin.