Neurofibromatosis-1 regulates mTOR-mediated astrocyte growth and glioma formation in a TSC/Rheb-independent manner

Abstract

Converging evidence from the analysis of human brain tumors and genetically engineered mice has revealed that the mammalian target of rapamycin (mTOR) pathway is a central regulator of glial and glioma cell growth. In this regard, mutational inactivation of neurofibromatosis-1 (NF1), tuberous sclerosis complex (TSC), and PTEN genes is associated with glioma formation, such that pharmacologic inhibition of mTOR signaling results in attenuated tumor growth. This shared dependence on mTOR suggests that PTEN and NF1 (neurofibromin) glial growth regulation requires TSC/Rheb (Ras homolog enriched in brain) control of mTOR function. In this report, we use a combination of genetic silencing in vitro and conditional mouse transgenesis approaches in vivo to demonstrate that neurofibromin regulates astrocyte cell growth and glioma formation in a TSC/Rheb-independent fashion. First, we show that Nf1 or Pten inactivation, but not Tsc1 loss or Rheb overexpression, increases astrocyte cell growth in vitro. Second, Nf1-deficient increased mTOR signaling and astrocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in glial progenitors of Nf1(+/-) mice does not lead to glioma formation. Collectively, these findings establish TSC/Rheb-independent mechanisms for mTOR-dependent glial cell growth control and gliomagenesis relevant to the design of therapies for individuals with glioma.

Fig. 1.

Neurofibromin and Pten loss in astrocytes results in increased mTOR-dependent proliferation. Whereas Nf1^−/− (A) and Pten^−/− (B) astrocytes exhibit increased mTOR activation (phospho-S6 Ser-240/244) and proliferation sensitive to 10 nM rapamycin inhibition (C and D), Tsc1-deficient (E) and Rheb-expressing (F) astrocytes exhibit robust mTOR activation but no increase in proliferation in vitro. Total S6 and α-tubulin were included as internal loading controls. Asterisks denote statistically significant differences (P ≤ 0.0001) between WT and Nf1^−/− or Pten^−/− astrocytes. Bars indicate SEM.

Fig. 2.

Nf1-deficient astrocyte hyperproliferation is not dependent on Rheb expression. (A) Convergence on mTOR pathway by neurofibromin, PTEN, TSC, and Rheb. (B) Rheb shRNA silencing reduces mTOR activation in Tsc1-deficient astrocytes (B; Right), but has no effect on mTOR activity in Nf1^−/− astrocytes (B; Left). Representative blots are shown. (C) Rheb shRNA knockdown in Nf1-deficient astrocytes does not reduce cell proliferation. Bars indicate SEM of six replicate wells per condition for the representative experiment shown in B (Left).

Fig. 3.

Conditional expression of Rheb in astrocytes does not increase cell growth in vitro. (A) Conditional targeting strategy for the generation of LSL-Rheb mice (line 13431). (B) Ad5 virus-mediated Cre delivery results in eGFP (Left; immunofluorescence microscopy) and Rheb (Right; Western blot) expression compared with LacZ delivery. Both eGFP and Rheb expression in Cre-transduced astrocytes is eliminated with doxycycline. (C) Cre-mediated Rheb expression results in increased mTOR signaling (phospho-S6 Ser-240/244 and Ser-235/236). (D) No change in astrocyte proliferation was observed following Cre-mediated Rheb expression as assessed by [³H]thymidine incorporation. Bars indicate SEM.

Fig. 4.

Conditional Rheb expression in astroglial cells results in increased glial numbers and proliferation in vivo. (A) No effect of Rheb expression on body size or brain weight was observed in Rheb^GFAP mice. (B) GFAP-Cre-mediated Rheb expression results in increased numbers of pS6 (Ser-240/244) cells, (C) Ki67⁺ proliferating cells, and (D) GFAP⁺ cells in vivo. Magnification = 10×. (Insets) Representative pS6⁺ (B) and Ki67⁺ (C) cells. (Scale bars, 100 μm.) Bars indicate SEM.

Fig. 5.

Conditional Rheb expression in Nf1^+/− mice is insufficient for glioma formation. (A) Robust eGFP expression is seen in the optic nerves of Rheb^GFAP mice. Magnification, 10×. (Scale bar, 100 μm.) (B) Nf1^+/−; Rheb^GFAP mice have normal-appearing optic nerves and no increase in optic nerve volume, whereas Nf1^+/−GFAPCKO mice develop prechiasmatic optic nerve and chiasmal gliomas. FF, Nf1^flox/flox(WT). Magnification, 10×. (Scale bar, 100 μm.) (C and D) Nf1^+/−; Rheb^GFAP mice have increased numbers of Ki67⁺ proliferating cells in the optic nerve but no focal areas of GFAP⁺ cells indicative of optic glioma, whereas Nf1^+/−GFAPCKO mice demonstrated both increased numbers of Ki67⁺ cells and GFAP immunostaining. Magnification, 20×. (Scale bars, 100 μm.) The arrows denote Ki67⁺ cells. Bars indicate SEM.