Articles

NF2/Merlin Is a Novel Negative Regulator of mTOR Complex 1, and Activation of mTORC1 Is Associated with Meningioma and Schwannoma Growth

Marianne F. James, Sangyeul Han, Carolyn Polizzano, Scott R. Plotkin, Brendan D. Manning, Anat O. Stemmer-Rachamimov, James F. Gusella, Vijaya Ramesh
DOI: 10.1128/MCB.01581-08

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Rapamycin inhibits the growth of primary merlin-deficient arachnoidal and meningioma cells. (A) Primary control (scr RNAi) and merlin knockdown (m5 RNAi) arachnoidal (AC030) cells were treated with or without rapamycin starting at day 2 postinfection with lentiviruses expressing shRNAs and every other day thereafter for 12 days. Phase-contrast images demonstrate an increase in the sizes of untreated (vehicle-treated) merlin RNAi arachnoidal cells compared to those of scr RNAi arachnoidal cells (original magnification, ×10). Rapamycin (1 and 20 nM) inhibits the increase in the sizes of merlin RNAi cells compared to those of vehicle-treated merlin RNAi arachnoidal cells. (B) Primary control and merlin knockdown arachnoidal (AC030) cells, treated with (+) or without (−) rapamycin (Rap; 10 nM) every other day for 10 days, were evaluated by FACS analyses to determine relative cell sizes. The x axes indicate relative cell sizes according to FSC-H values. (Left) Merlin m5 RNAi increases the sizes of arachnoidal cells, as determined by the rightward shift in the mean FSC-H histogram compared to that for control scr-treated arachnoidal cells. (Right) Rapamycin abolishes the effect of merlin m5 RNAi on cell size. (C) The enlargement of cells in primary merlin-deficient MN304 meningioma cultures treated with vehicle only is inhibited by treatment with rapamycin (10 nM) every other day for 7 days (original magnification, ×10).

  • FIG. 2.
    FIG. 2.

    The mTORC1 pathway is aberrantly activated in merlin-deficient NF2 target cell types in a growth factor-independent manner but is sensitive to nutrient deprivation. (A) Three primary arachnoidal (AC028, AC030, and AC032) and three primary merlin-deficient [merlin(−)] meningioma (MN380, MN383, and MN302) cell lines were deprived of serum (in 0.2% FBS) overnight prior to being harvested to obtain protein lysates. S6, a common marker of mTORC1 activation, exhibits elevated phosphorylation in merlin-deficient MN cells compared to that in normal arachnoidal cells, with no change in total S6 protein levels. GAPDH protein levels demonstrate equivalent sample loading. p-S6 (S235/236), S6 phosphorylated at S235/236. (B) Arachnoidal cells stably expressing three different lentivirus-mediated shRNAs (m5, m6, and m8) targeting merlin exhibit increased phosphorylation of mTOR (S2448) and mTORC1 effectors p70-S6K (at Thr389) and S6 (at S240/244 and S235/236), as well as cyclin D1, a downstream target of mTORC1, upon merlin knockdown compared to that in control (scr RNAi) cells under conditions of serum deprivation (in 0.2% FBS). Total levels of mTOR, S6, and S6K proteins were unchanged. p-mTOR (S2448), mTOR phosphorylated at S2448; p-S6K (T389), S6K phosphorylated at T389; p-S6 (S240/244) and p-S6 (S235/236), S6 phosphorylated at S240/244 and S235/236, respectively. (C) Merlin knockdown in arachnoidal cells (by m5, m6, and m8) results in increased accumulation of phosphorylated species of 4EBP1 compared to that in scr control RNAi cells under conditions of serum deprivation (in 0.2% FBS). 4EBP1 phosphorylation (detected by anti-4EBP1 antibody) is reflected by 4EBP1 electrophoretic mobility results. (D) Cell cycle evaluation by FACS analysis demonstrates increased sizes of merlin knockdown cells (expressing m5 and m8 shRNAs) compared to those of scr control cells in G1 and G2/M phases of the cell cycle. (E) To examine effects of merlin deficiency on nutrient deprivation, control (scr) and merlin RNAi (m5) knockdown arachnoidal cells were grown overnight with full serum (15% FBS) prior to being shifted into Dulbecco's PBS for 0, 20, 40, or 60 min. The phosphorylation of S6 (at S240/244) is appropriately terminated in merlin RNAi arachnoidal cells, similar to that in control scr cells. − aa, amino acid deprivation.

  • FIG. 3.
    FIG. 3.

    Merlin-deficient meningiomas and vestibular schwannomas exhibit constitutive S6 phosphorylation. Patient-derived merlin-deficient meningioma (MN258−/− and MN336−/−) and vestibular schwannoma (xT1640) tissues were stained immunohistochemically to detect S6 phosphorylation (at S240/244 and S235/236). Data are representative of results for four benign merlin-deficient meningiomas and five benign vestibular schwannomas. Sections of normal nerve tissue (negative control) did not exhibit S6 phosphorylation (original magnification, ×40).

  • FIG. 4.
    FIG. 4.

    Merlin-deficient mTORC1 activation is PI3K-Akt and MAPK/ERK independent. (A and B) Merlin-deficient meningioma [merlin(−) MN] (A) and merlin knockdown (m5, m6, or m8 RNAi) (B) arachnoidal cells exhibit elevated ERK1/ERK2 phosphorylation compared to that in control (scr RNAi) arachnoidal cells. In contrast, merlin-deficient target cells exhibit no change in the phosphorylation of Akt (A and B) or PRAS40 (B), a substrate of Akt and a negative regulator of mTORC1, compared to that in control arachnoidal cells. Cells were treated as described in the legend to Fig. 2A and B. (C to F) Control (scr RNAi) and merlin knockdown (m5 RNAi) arachnoidal cells were deprived of serum (in 0.2% FBS) overnight, and the merlin knockdown cells were then incubated with the indicated concentrations of rapamycin (Rap), LY294002, wortmannin (Wm), and UO126 for 30 min. Cell lysates were assayed for S6 phosphorylation by immunoblot analyses. −, absent. (C) The mTORC1-specific inhibitor rapamycin blocks constitutive S6 activation. (D) The PI3K inhibitor LY294002 blocks S6 activation but does not affect MAPK/ERK signaling. (E, top) Wortmannin does not block S6 activation, indicating that mTORC1 activation is independent of PI3K-Akt signaling. (Bottom) Wortmannin potency is demonstrated by the inhibition of IGF1-stimulated Akt phosphorylation in arachnoidal cells pretreated with wortmannin (100 nM), in contrast to that in untreated cells. (F) U0126, a MEK1/MEK2 inhibitor, blocks ERK1/ERK2 phosphorylation but has no effect on S6 phosphorylation.

  • FIG. 5.
    FIG. 5.

    Merlin expression inhibits mTORC1 signaling. (A) HEK293T cells were cotransfected with HA-S6K and vector only (V) or FLAG-tagged Rheb, TSC1-TSC2, or NF2 protein isoform 1 (NF2 iso 1) or isoform 2 (NF2 iso 2). After 24 h, cells were subjected to serum starvation (0% FBS) overnight and stimulated with insulin (150 nM) for 15 min. Both NF2 protein isoforms inhibit S6K phosphorylation compared to that in cells expressing the vector only. Rheb expression activates and TSC1-TSC2 expression inhibits the mTORC1 pathway. (B) HEK293T cells were cotransfected with HA-S6K and vector only or FLAG-tagged WT NF2 protein or mutant NF2 protein constructs expressing L64P, S518A, or S518D. Similar to WT NF2 protein, NF2 mutant proteins expressing S518A and S518D inhibit S6K activation in full-growth medium (10% FBS), but a patient-derived mutant (L64P) protein does not. p-NF2 (S518), NF2 protein phosphorylated at S518. (C) Merlin-deficient meningioma [merlin(−) MN] cells were transduced with vector only or NF2 protein (MOI, 50) and subjected to serum starvation (0% FBS) overnight. NF2 protein-infected cells exhibit the inhibition of S6 phosphorylation compared to that in vector-infected cells. (D) HEK293T cells were cotransfected with HA-S6K and vector only or FLAG-NF2 protein, TSC2 siRNA (TSC2 si), or FLAG-NF2 protein and TSC2 siRNAs together (NF2 + TSC si). Immunoblot analyses of HA immunoprecipitations (IP) using an anti-HA antibody indicate that S6K phosphorylation is not inhibited by NF2 protein in TSC2 siRNA-expressing cells in full-growth medium. The decrease in levels of S6K phosphorylated at T389 in the NF2 + TSC2 si lane compared to those in the TSC2 si lane is due to reduced levels of HA-S6K expression. (E) Tsc2−/− MEFs were infected with vector (MOI, 50) or NF2 protein (MOIs, 50 and 100) as described in the legend to panel C. NF2 protein does not inhibit S6K or S6 phosphorylation compared to that in vector-infected cells. Lanes −, untransfected cells. (F) Among HEK293T cells transfected as described in the legend to panel D, those coexpressing NF2 protein and Rheb show no inhibition of S6K phosphorylation.

  • FIG. 6.
    FIG. 6.

    Akt signaling is attenuated in cells lacking NF2 protein due to mTORC1-mediated negative feedback inhibition. (A) Control (scr RNAi) and merlin knockdown (m5 RNAi) arachnoidal cells were deprived of serum (in 0.2% FBS) overnight and stimulated with insulin (100 nM) for the times indicated. scr RNAi arachnoidal cells exhibit a time-dependent increase in activated Akt (S473) and S6 (S235/236) phosphorylation, in contrast to merlin knockdown arachnoidal cells, which exhibit impaired Akt phosphorylation and constitutive S6 activation that is insensitive to insulin stimulation. (B) Merlin-deficient meningioma [merlin(−) MN] cells and control (scr) and merlin (m5) RNAi arachnoidal cells were incubated with (+) and without (−) rapamycin (Rap; 10 nM) for 24 h. Untreated cells in complete medium (15% FBS) exhibit S6 phosphorylation (at S240/244) and the attenuation of Akt phosphorylation (at S473), resulting from constitutive activation of an mTORC1-S6K negative feedback loop. In the presence of rapamycin, mTORC1-S6K signaling is blocked, preventing S6 phosphorylation, and Akt phosphorylation is restored by relief from negative feedback. Under serum-free (SF; 0% FBS) conditions, merlin-deficient meningioma cells do not exhibit the restoration of Akt signaling. In addition, cyclin D1 levels are reduced in rapamycin-treated cells, suggesting that expression is, in part, mTORC1 dependent.

  • FIG. 7.
    FIG. 7.

    Nf2-deficient (Nf2−/−) MEFs exhibit constitutive mTORC1 activation compared to Nf2 WT (Nf2+/+) MEFs. (A) Nf2−/− MEFs, plated confluently and subconfluently (subconfl), exhibit constitutive S6 activation, in contrast to Nf2+/+ MEFs, under conditions of serum starvation (0% FBS). (B) Nf2−/− MEFs and Tsc2−/− MEFs exhibit increased accumulation of phosphorylated species of 4EBP1 compared to that in control Nf2+/+ and Tsc2+/+ MEFs, respectively, under conditions of serum starvation (0% FBS). 4EBP1 phosphorylation (detected by anti-4EBP1 antibody) is reflected by 4EBP1 electrophoretic mobility results. (C) Rapamycin (Rap) suppresses the proliferation of Nf2−/− MEF cells. Nf2+/+ and Nf2−/− MEFs incubated with 20 nM rapamycin or vehicle (DMSO) for 24 h were examined for S-phase entry by using BrdU incorporation as detailed in Materials and Methods. Data are presented as mean percentages of DAPI-stained cells ± standard errors of the means. *, P < 0.01 for DMSO-treated control versus rapamycin-treated groups or Nf2+/+ versus Nf2−/− MEF cells (n = 5); +, present; −, absent.

Additional Files

  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Fig. S1 (Rapamycin inhibits growth of merlin knockdown arachnoidal cells).
      TIF file, 1.46 MB.
    • Supplemental file 2 - Fig. S2 (Merlin-deficient meningioma cell mTORC1 signaling in the absence of mitogenic input).
      TIF file, 466K.
    • Supplemental file 3 - Fig. S3 (Merlin knockdown arachnoidal cell growth factor receptor expression or activation).
      TIF file, 674K.
    • Supplemental file 4 - Supplemental methods.
      TIF file, 369K.
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KEYWORDS

cell proliferation
Neurofibromin 2
transcription factors

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