Neurofibromatosis-1 Regulates Mtor-Mediated Astrocyte Growth

Neurofibromatosis-1 regulates mTOR-mediated astrocyte growth and glioma formation in a TSC/Rheb-independent manner Sutapa Banerjee, Nikkilina R. Crouse, Ryan J. Emnett, Scott M. Gianino, and David H. Gutmann1 Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110 Edited* by Webster K. Cavenee, Ludwig Institute, University of California at San Diego, La Jolla, CA, and approved August 15, 2011 (received for review December 17, 2010) Converging evidence from the analysis of human brain tumors seizures and death, whereas Nf1 loss in these same neuroglial and genetically engineered mice has revealed that the mamma- progenitors has no effect on mouse viability or epileptogenesis (16, lian target of rapamycin (mTOR) pathway is a central regulator of 17). Importantly, although Pten-, hamartin-, and neurofibromin- glial and glioma cell growth. In this regard, mutational inactiva- deficient glial phenotypes have different biological effects, each tion of neurofibromatosis-1 (NF1), tuberous sclerosis complex is completely inhibited by rapamycin in vitro and in vivo (15, 18– (TSC), and PTEN genes is associated with glioma formation, such 21). Collectively, these observations support a model in which fi that pharmacologic inhibition of mTOR signaling results in atten- neuro bromin-regulated mTOR-dependent growth regulation uated tumor growth. This shared dependence on mTOR suggests is distinct from TSC/Rheb-mediated mTOR-dependent growth that PTEN and NF1 (neurofibromin) glial growth regulation regulation in the brain. To critically examine the requirement for the TSC/Rheb axis requires TSC/Rheb (Ras homolog enriched in brain) control of fi mTOR function. In this report, we use a combination of genetic in neuro bromin mTOR-dependent growth regulation in the silencing in vitro and conditional mouse transgenesis approaches brain, we used a combination of pharmacologic and genetic silencing approaches in vitro coupled with conditional knockout in vivo to demonstrate that neurofibromin regulates astrocyte and overexpression transgenesis strategies in vivo. We show that cell growth and glioma formation in a TSC/Rheb-independent fi Nf1 Pten neuro bromin regulation of glial cell growth and glioma for- fashion. First, we show that or inactivation, but not mation is TSC/Rheb-independent, and that the functional con- Tsc1 loss or Rheb overexpression, increases astrocyte cell growth Nf1 fi sequences of TSC/Rheb mTOR regulation in astroglial cells are in vitro. Second, -de cient increased mTOR signaling and as- distinct from those conferred by Nf1 gene inactivation. trocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in Results Nf1+/− glial progenitors of mice does not lead to glioma forma- mTOR Activation Differentially Regulates Cell Growth in Astrocytes tion. Collectively, these findings establish TSC/Rheb-independent in Vitro. Previous studies have demonstrated that Nf1, Pten, and mechanisms for mTOR-dependent glial cell growth control and Tsc1 gene inactivation each results in increased mTOR activa- gliomagenesis relevant to the design of therapies for individuals tion in astrocytes in vitro (15, 19, 20). In addition, Rheb over- with glioma. expression to mimic Tsc1/Tsc2 gene inactivation similarly increases mTOR activation (15). In each case, this mTOR acti- astrocytoma | optic glioma | glia vation is completely inhibited by rapamycin treatment (Fig. 1), suggesting that mTOR signaling is a critical regulator of cell utations in several tumor suppressor genes and receptor growth in astrocytes. However, mTOR activation as a conse- Mtyrosine kinases (RTKs) deregulate cell growth and survival quence of Nf1 and Pten loss results in increased astrocyte pro- by increasing mammalian target of rapamycin (mTOR) signaling. liferation in vitro, whereas increased mTOR signaling following This convergence on mTOR is well-illustrated in brain tumors Tsc1 loss, Tsc2 loss, or Rheb overexpression (>300-fold) has no (gliomas) in which mutations lead to mTOR-dependent growth such effect. This differential effect does not reflect the magni- deregulation. In this regard, copy-number increases or gain-of- tude of mTOR activation as measured by S6 phosphorylation on function mutations in key RTK molecules (PDGFR, EGFR, and Ser residues 240/244 (Fig. 1 and Fig. S1). Moreover, the differ- ERBB2) enhance glioma growth by activating mTOR (1). Simi- ence between TSC/Rheb and neurofibromin/PTEN mTOR reg- PTEN ulation of astrocyte proliferation is not due to the activation of larly, loss-of-function mutations in the , tuberous sclerosis − − − − complex (TSC), and neurofibromatosis-1 (NF1) tumor suppres- other signaling pathways, as both Nf1 / and Pten / astrocyte sor genes result in glioma formation, which is reduced by phar- hyperproliferation is abolished following mTOR inhibition. To- macologic mTOR inhibition. The shared activation of mTOR by gether, these findings suggest that mTOR regulation by neuro- PTEN, TSC (tuberin and hamartin), and NF1 (neurofibromin) fibromin/PTEN is distinct from TSC/Rheb (Fig. 2A). loss has suggested a model in which protein kinase B (PKB)/AKT hyperactivation following PTEN or neurofibromin loss leads to Neurofibromin Growth Regulation of Astrocyte Proliferation Does tuberin phosphorylation (2–5), loss of tuberin/hamartin complex Not Depend on Rheb in Vitro. Prior analyses of neurofibromin inhibition of Ras homolog enriched in brain (Rheb) activity (6– mTOR growth control suggested that the Akt hyperactivation 10), and Rheb-mediated mTOR activation (11–14) relevant to resulting from Nf1 inactivation (Fig. S2A) leads to tuberin glioma growth and targeted therapy. However, several experimental findings call this unified TSC/ Rheb model of mTOR regulation into question. First, although Author contributions: S.B. and N.R.C. designed research; S.B., N.R.C., R.J.E., and S.M.G. Nf1 and Pten inactivation cause increased astroglial cell growth performed research; S.B. and N.R.C. analyzed data; and S.B., N.R.C., and D.H.G. wrote the in vitro, no such increase in proliferation is observed following paper. Tsc1 loss or Rheb overexpression (15). Second, Tsc1 loss in The authors declare no conflict of interest. astrocytes results in increased cell size in the brain, whereas Nf1 *This Direct Submission article had a prearranged editor. inactivation in vitro or using the same Cre driver mouse strain 1To whom correspondence should be addressed. E-mail: [email protected]. Tsc1 in vivo has no discernable effect on cell size (16, 17). Third, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inactivation in neuroglial progenitor cells results in intractable 1073/pnas.1019012108/-/DCSupplemental. 15996–16001 | PNAS | September 20, 2011 | vol. 108 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1019012108 Downloaded by guest on September 30, 2021 Fig. 1. Neurofibromin and Pten loss in astrocytes results in increased mTOR- dependent proliferation. Whereas − − − − Nf1 / (A)andPten / (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)astrocytesex- hibit robust mTOR activation but no increase in proliferation in vitro. Total S6 and α-tubulin were included as internal loading controls. Asterisks de- note statistically significant differences − − (P ≤ 0.0001) between WT and Nf1 / or − − Pten / astrocytes. Bars indicate SEM. − − phosphorylation and inactivation (21). First, we observed no Rheb in Nf1 / astrocytes (Fig. 2B and Fig. S2D). In contrast, change in tuberin phosphorylation (residues Thr-1462 and Ser- S6 phosphorylation is attenuated by Rheb silencing in Tsc1- − − 969) in Nf1 / astrocytes relative to wild-type astrocytes (Fig. deficient astrocytes. Third, following Rheb knockdown in Nf1- S2B). Similarly, we observed no change in tuberin Ser-969 fi −/− de cient or WT astrocytes, we observed no reproducible changes phosphorylation in Pten astrocytes compared with wild-type in proliferation (Fig. 2C and Fig. S2 E and F). Collectively, these astrocytes (Fig. S2C). Second, we found no change in S6 phos- observations demonstrate that neurofibromin regulation of as- phorylation following small hairpin RNA (shRNA) silencing of trocyte mTOR signaling and proliferation is not dependent on TSC/Rheb function. Conditional Rheb Activation in Mice Results in Increased Astrocyte mTOR Signaling and Proliferation in Vivo. To extend the above in vitro studies, we next generated conditional Rheb-expressing mice (Fig. 3A). The conditional Rheb transgene was engineered into the Rosa locus by homologous recombination, resulting in two independent strains of LSL-Rheb mice (lines 13431 and 13433). In addition, the transgene contained a reverse tetracy- cline control element, allowing doxycycline regulation of Rheb expression as well as a downstream enhanced green fluorescent protein (eGFP) driven from an internal ribosomal entry site (IRES). We verified the inducible and regulatable expression of Rheb in primary forebrain astrocytes from these mice. Following infection with Ad5-Cre virus, robust GFP and Rheb expression was observed, which was abrogated following doxycycline treatment (line 13431; Fig. 3B). No GFP or Rheb expression was observed in identical astrocytes infected with Ad5-LacZ. Similar results were also obtained using line 13433 (Fig. S3). Moreover, as seen following viral Rheb expression (Fig. 1F), transgenic expression

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