In Vivo Regulation of TGF-b by R-Ras2 Revealed

Published OnlineFirst August 23, 2012; DOI: 10.1158/0008-5472.CAN-12-1972

Cancer Research

Molecular and Cellular Pathobiology

In Vivo Regulation of TGF-b by R-Ras2 Revealed through Loss of the RasGAP Protein NF1 Deanna M. Patmore1, Sara Welch1, Patricia C. Fulkerson1, Jianqiang Wu1, Kwangmin Choi1, David Eaves2, Jennifer J. Kordich1, Margaret H. Collins3, Timothy P. Cripe2, and Nancy Ratner1

Abstract Ras superfamily proteins participate in TGF-b—mediated developmental pathways that promote either tumor suppression or progression. However, the specific Ras proteins, which integrate in vivo with TGF-b signaling pathways, are unknown. As a general approach to this question, we activated all Ras proteins in vivo by genetic deletion of the RasGAP protein Nf1 and examined mice doubly deficient in a Ras protein to determine its requirement in formation of TGF-b—dependent neurofibromas that arise in Nf1-deficient mice. Animals lacking Nf1 and the Ras-related protein R-Ras2/TC21 displayed a delay in formation of neurofibromas but an acceleration in formation of brain tumors and sarcomas. Loss of R-Ras2 was associated with elevated expression of TGF-b in Nf1-deficient Schwann cell precursors, blockade of a Nf1/TGFbRII/AKT-dependent autocrine survival loop in tumor precursor cells, and decreased precursor cell numbers. Furthermore, the increase in size of sarcomas from xenografts doubly deficient in these genes was also found to be TGF-b—dependent, in this case resulting from cell nonautonomous effects on endothelial cells and myofibroblasts. Extending these findings in clinical specimens, we documented an increase in TGF-b ligands and an absence of TGF-b receptor II in malignant peripheral nerve sheath tumors, which correspond to tumors in the Nf1-deficient mouse model. Together, our findings reveal R-Ras2 as a critical regulator of TGF-b signaling in vivo. Cancer Res; 72(20); 5317–27. 2012 AACR.

Introduction Ras proteins are molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form and signal through effector pathways including Ral, phosphoinositide 3-kinase (PI3K)-AKT and Raf-MEK-ERK to regulate proliferation, cell death, and cell differentiation (1, 2). Individual members of the Ras superfamily can have unique roles in diverse cell compartments (3, 4). The commonly studied Ras oncogenic proteins encoded by the H-Ras, K-Ras, and N-Ras genes (Ras) are activated by mutation in up to 50% of human cancers (5). The related R-Ras family, encoded by the R-Ras, TC21/R-Ras2 (subsequently TC21), and R-Ras3/M-Ras genes, also has oncogenic potential (6). Here we focus on TC21, a transforming oncogene mutated in human tumor cell lines (7) and shown to induce lymphoma in vivo (8), in the context of the NF1 Ras GTPase-activating protein (GAP). Ras signaling is inactivated by GAPs, including the NF1 tumor suppressor protein neurofibromin. Neurofibromin is Authors' Affiliations: 1Divisions of Experimental Hematology and Cancer Biology; 2Hematology and Oncology, and 3Pathology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Nancy Ratner, Division of Experimental Hematology, Children's Hospital Research Foundation, 3333 Burnet Avenue, MLC 7013, Cincinnati, OH 45229-3039. Phone: 513-636-9469; Fax: 513636-3549; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-1972 2012 American Association for Cancer Research.

a GAP for Ras proteins (9), so that sustained activation of each expressed Ras protein is predicted in cells that rely on neurofibromin function. Mutations in the NF1 gene result in neurofibromatosis type 1 (NF1; 10). NF1 patients develop disfiguring benign peripheral nerve tumors, neurofibromas; plexiform neurofibroma can transform into sarcomas known as malignant peripheral nerve sheath tumors (MPNST), a leading cause of death in adults with NF1 (11). We reasoned that specific roles of individual Ras proteins would be revealed in the setting of NF1 loss in peripheral nerve cells. Consistent with this idea, increased migration of Nf1/ Schwann cells was rescued by a dominant negative allele of TC21 (12). Ras signaling is critical to activate a variety of downstream kinase cascades. Although mitogen-activated protein kinases (MAPK) Erk1/2, c-Jun N-terminal kinase, and p38SAPK MAPK act as downstream effectors of TC21 in certain cell lines (6), PI3K-AKT is currently believed to be a major TC21 effector (13). A recent in vivo study implicates TC21 in PI3K signaling downstream of the antigen receptor in T cells (14). In addition to PI3K signaling, TC21 was recently linked to TGF-b in vitro; overexpression of an activated TC21 allele caused cells to lose responsiveness to the growth inhibitory effects of TGF-b (13). Interestingly, TGF-b proteins can act as either tumor suppressors or promoters (15, 16). TGF-b ligands 1, 2, and 3 regulate cell proliferation, death, and differentiation through interaction with receptors TGFbRI (ALK5) and TGFbRII, and the TGFbRIII coreceptor (17). Downstream of TGF-b receptors (TGFbR), activation of SMAD proteins and PI3K-AKT signaling are thought to control TGF-b tumor suppression, although activation of RhoA, TAK1, and PI3K-AKT are implicated in

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TGF-b oncogenesis (16). Decreased or altered TGF-b responsiveness and increased expression or activation of TGF-b ligands is common as tumors progress. For example, TGF-b can promote tumorigenesis through sequestration of mutant p53 (18), and have nonautonomous effects on tumor stroma (19). We identify TC21 as a regulator of TGF-b function in vivo. Previous studies examined cross talk between the Ras and TGFb—signaling pathways, mainly in vitro (20–22). Ras/MAPK activates the TGF-b promoter (23, 24), and Ras/Erk signaling blocks SMAD translocation to the nucleus through phosphorylation of SMAD2/3 (20). In vivo, RAS/MAPK can phosphorylate p53, which then interacts with SMAD proteins (25). Neurofibromas and MPNSTs derive from neural crest lineage cells, more mature Schwann cell precursors (SCP), and/or differentiated mature Schwann cells (26). To study development, we crossed TC21-deficient mice (TC21/; ref. 14) to Nf1þ/ mice (27). To study tumorigenesis, we crossed TC21/ mice to Nf1fl/fl;DhhCre mice that form neurofibromas, and Nf1þ/;Trp53þ/ (NPCis) mice that serve as a model of soft tissue sarcomas and brain tumors with histology of glioblastoma (28), and used a xenograft model of human MPNST. We found that Nf1 mutation renders SCPs insensitive to TGF-b—mediated cell death and define a TGF-b autocrine survival loop, correlating with benign neurofibroma formation. Nf1 mutants lacking TC21 restore TGF-b sensitivity and benign tumorigenesis is delayed. Conversely, loss of TC21 increases TGF-b—induced malignancy in the NPCis model. In NF1 MPNST xenografts, loss of TC21 accelerates tumor growth in a noncell autonomous manner. In summary, TC21 is a major regulator of TGF-b production that functions in development and tumorigenesis in vivo.

Materials and Methods Mice We housed mice in a temperature- and humidity-controlled vivarium on a 12-hour dark–light cycle with free access to food and water. TC21þ/ and R-Rasþ/ mice were obtained on C57Bl/6/129 mixed background after 4 generations of backcross onto C57/Bl6 (14), then bred to homozygosity. We mated TC21/ and R-Ras/ mice to Nf1þ/ C57/Bl6 mice (27) to obtain TC21/;Nf1þ/ and R-Ras/;Nf1þ/ mice, which were intercrossed to obtain mutant embryos. TC21/ mice were mated to Nf1fl/fl;DhhCre mice (26) to obtain TC21þ/;Nf1fl/þ; DhhCre mice. F1 mice were intercrossed to obtain TC21/; Nf1fl/fl;DhhCre mice and TC21/;Nf1fl/þ;DhhCre littermates. We bred TC21/ mice to NPCis C57BL/6 mice (28) to obtain TC21þ/;NPCis mice. These mice were mated with TC21/ to obtain TC21/;NPCis mice. TC21þ/;NPCis littermates were bred to C57BL/6 wild-type mice for parallel controls. For tumor experiments we analyzed male mice. Cell culture We dissociated dorsal root ganglia (DRG) from E12.5 embryos and plated cells in serum-free medium (29). We used cells direct from embryos for precursor numbers. At passage 2 to 3, 500 cells/well were plated then inhibitors, antibody or lentivirus added after 24 hours. Spheres were counted 3 days later.

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Each experiment shown represents 3 or more independent experiments. Anti-TGF-b antibody, rhTGFb1, and rabbit immunoglobulin G (IgG) were from R&D Systems. TGFbR1, MEK1 (Cayman Chemicals), AKT (Selleck Chemicals), p38SAPK, and ROCK (Calbiochem) inhibitors were dissolved in dimethyl sulfoxide. MPNST cell lines included 26T, T265, 8814 [WT for p53 (30)] and S462TY [derived from the S462 cell line by 2 rounds of in vivo growth as xenografts (31)]. Cells were maintained in Dulbecco's Modified Eagle's Medium/10% FBS/1% penicillin/streptomycin. Immunohistochemistry and histology Six-micrometer formalin-fixed paraffin sections or 12-mm 4% paraformaldehyde-fixed frozen sections were stained with phospho-SMAD2/3, phospho-AKT (Cell Signaling), SMA (Dako), antineurofilament, and meca-32 (DSHB). Secondary incubations used host-appropriate secondary antibodies. Neurofibroma sections were submitted to the CCHMC Pathology Laboratory for hematoxylin and eosin stain (H&E), toluidine blue, and rabbit polyclonal anticow S100b (Dako). Western blot analysis We lysed cells, tissue, and tumor sections as described (32). We separated proteins on 4% to 20% TrisHCl acrylamide gels (Biorad), transferred to polyvinylidene difluoride membranes (Millipore), and probed membranes with: antiTC21 (Abnova), phospho-AKT, phospho-SMAD2/3, phospho-42/44-MAPK, and b-actin (Cell Signaling). Detection used horseradish peroxidase-conjugated secondary antibodies (BioRad) and ECL Plus developing system (Amersham Biosciences). qRT-PCR We isolated total RNA with an RNeasy kit (Qiagen) and carried out cDNA synthesis (Invitrogen Superscript III). We used triplicate reactions to conduct quantitative real-time PCR (qRT-PCR; ABI 7500-Sequence Detection System; 32). Values for genes of interest were normalized to glyceraldehyde-3phosphate dehydrogenase (mouse samples) or b-actin (human samples) and fold change calculated by the DDct method. Lentiviral infection We infected MPNST cells at 50% confluence with TRIPZ shTC21 or nontarget control (Open Biosystems). We incubated lentiviral particles with MPNST cells in the presence of polybrene (8 mg/mL; Sigma-Aldrich) daily for 3 days, followed by selection in puromycin (2.5 mg/mL; Sigma-Aldrich) then maintained cells in media containing puromycin; 2 mg/mL doxycycline (MP Biomedicals)-induced shRNA expression. Mouse xenograft We injected 2.3 106 S462TY cells in 150 mL with 30% matrigel (BD Biosciences) into flanks of 5-/6-week-old female athymic nude (nu/nu) mice (Harlan). We maintained mice on 1,875 ppm doxycycline feed (Test Diet). Tumor volumes and weight were measured twice weekly. We sacrificed mice before tumor size reached 10% body weight. For anti-TGF-b

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TC21 and TGF-b in NF1 Tumors

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Figure 1. Loss of TC21 extends survival of neurofibroma-bearing mice but decreases survival of NPCis mice. Kaplan–Meyer survival curves for Nf1 fl/fl / mice (A; log-rank test, p < 0.0001) and NPCis and NPCis;TC21/ mice (B; log-rank test, p ¼ 0.0001). and Nf1 ;DhhCre;TC21

treatment, we injected mice intraperitoneally with 3 mg/kg of IgG or anti-TGF-b antibody every 2 weeks. We dissected tumors; we fixed tumors in 4% paraformaldehyde or flashfroze and stored tumors at 80 C. Gene expression analysis All samples except for normal nerves were previously described (33). Affymetrix probes were remapped to RefSeq genes (version 11.0.1). Comparisons and data visualization were conducted using GeneSpring GX v7.3.1 (Agilent Technologies).

Results Loss of TC21 extends neurofibroma-bearing mice survival but decreases NPCis mice survival To define roles for TC21, we used TC21/ mice (Supplementary Fig. S1A). TC21 mRNA was less than 10-fold and protein expression lost (Supplementary Fig. S1B and S1C), confirming that the TC21 mutation is a null allele (14). Nf1/ embryos die by embryonic day 12.5 (E12.5; 27), but 90% of TC21/;Nf1/ embryos survived to E14.5 and 10% survived to E16.5 (Supplementary Table S1). Partial rescue of embryo viability was not observed in R-Ras/;Nf1/ embryos (Supplementary Table S1). Thus, TC21 plays a role in Nf1 embryonic development. To determine TC21 relevance to tumorigenesis, we generated Nf1fl/fl;DhhCre;TC21/ and NPCis;TC21/ mice. Loss of TC21 significantly extended survival in Nf1fl/fl;DhhCre mice (Fig. 1A). Nf1fl/fl;DhhCre;TC21/(n ¼ 15) mice survived up to 20 months while littermate controls died by 15 months. Mice required sacrifice because of morbidity secondary to paralysis that correlated with neurofibroma formation and spinal cord compression. A second cohort of mice (n ¼ 15) showed identical results (not shown). To test for effects of TC21 loss in malignancy, we generated NPCis;TC21/ mice. NPCis;TC21/ (n ¼ 13) mice died by 7 months whereas littermate controls (n ¼ 7) survived up to 13 months (Fig. 1B). Therefore, loss of TC21 in benign tumors extends survival, although paradoxically in a model of aggressive tumors, loss of TC21 decreases survival. In addition, NPCis;TC21/ mice died early because of


;DhhCre

rapid formation of aggressive brain tumors (Supplementary Fig. S2). A role for TC21 in tumor initiation Grade 1 genetically engineered mice (GEM) neurofibromas from Nf1fl/fl;DhhCre mice with or without TC21 did not differ in histology on H&E staining, or anti-S100b staining to mark Schwann cells (34; Fig. 2A). Toluidine blue þ metachromatic mast cells increased slightly in the absence of TC21 (not shown). At the time of sacrifice there was no difference in number or size (diameter) of neurofibromas in the 2 strains of mice (Fig. 2B and C). To explain how loss of TC21 extends survival in Nf1fl/fl; DhhCre mice we postulated that TC21 diminishes numbers of neurofibroma-initiating or sustaining multipotent selfrenewing cells (29). To test this, we used an in vitro model. SCPs from Nf1/ DRG cells give rise to more spheres than do wild-type (WT) or Nf1þ/ DRG cells, and Nf1/ DRG sphere cells form neurofibroma-like lesions upon xenotransplantation (29). Nf1/ DRGs formed significantly more primary spheres at clonal density than cells from WT DRG. Importantly, TC21/;Nf1/ DRG cells formed WT levels of spheres (Fig. 2D) consistent with a role of TC21 regulating numbers of tumor-initiating cells early in Nf1 tumorigenesis. A TGF-b autocrine loop in Nf1/ SCPs Because TGF-b proteins can act as tumor suppressors or tumor promoters and TC21 acts similarly (Fig. 1), and because TC21 and TGF-b have been linked in vitro (13), we examined TGF-b signaling in the context of Nf1 and TC21 null alleles. Cells from secondary spheres were plated at clonal density and tested for response to TGF-b1. WT SCP numbers were reduced by exposure to TGF-b1 in a dose-dependent manner. WT SCP cells died but Nf1/ SCPs did not die when exposed to TGF-b1 (Supplementary Fig. S3A). Loss of TC21 restored sensitivity to TGF-b1 (Fig. 3A). Nf1/ SCPs had 20-fold higher levels of TGF-b1 mRNA and protein compared with WT precursors (Fig. 3B and C) and loss of TC21 rescued TGF-b1 levels in Nf1/ spheres. TGF-b produced by Nf1/ spheres was functional, as Nf1/ SCPs plated at clonal density with function blocking anti-TGF-b



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Figure 2. A role for TC21 in tumor initiation. A, neurofibroma sections stained with H&E, anti-S100b antibody (brown), and toluidine blue for mast cell infiltration (arrows). Scale bar, 50 mm; Tol., toluidine. B, quantification of average tumor numbers/mouse. C, quantification of tumor size (diameter, mm). D, number of primary spheres from E12.5 DRG plated at clonal density.

antibody died but spheres in control IgG formed healthy spheres (Fig. 3D and E). In contrast, treatment of WT or TC21/;Nf1/ SCPs with anti-TGF-b antibody did not alter sphere numbers (Fig. 4A). Production of TGF-b and effects of function blocking antibody suggest that Nf1/ SCPs secrete TGF-b, enhancing their survival. The data support the hypothesis that Nf1/ SCPs are dependent on TGF-b for survival. In contrast, TGF-b1 mRNA was only slightly elevated in mature Schwann cells in sciatic nerves from Nf1fl/fl;DhhCre mice compared with WT sciatic nerve (2-fold change; Supplementary Fig. S3B). There was no significant difference in TGF-b1 levels between neurofibromas from Nf1fl/fl; DhhCre (n ¼ 8) and Nf1fl/fl;DhhCre;TC21/ (n ¼ 10) mice (Supplementary Fig. S3C). That elevation and rescue of TGF-b1 mRNA expression levels in the TC21/;Nf1/ setting is most pronounced early in Schwann cell development suggests that TC21 activation plays roles in SCP viability and growth, not neurofibroma growth. The mechanism(s) by which TC21 loss enhances TGF-b expression may include alterations in signaling pathways that change in cell maturation and/or alterations because of as-yetunidentified mutations in neurofibroma Schwann cells during tumor formation. Survival of Nf1/ SCPs requires TGF-b and AKT To determine how TGF-b affects survival of Nf1/ SCPs, we used specific inhibitors. Treatment of Nf1/ SCPs with a

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TGFbR1 inhibitor blocked sphere formation but did not affect WT or TC21/;Nf1/ spheres (Fig. 4B). PI3K/AKT has been implicated downstream of TGF-b signaling, and treatment with an AKT inhibitor significantly reduced Nf1/ sphere formation (Fig. 4C). In contrast, MEK1, ROCK, and p38SAPK inhibitors had no effect on Nf1/ sphere formation (Supplementary Fig. S4A). Efficacy of inhibitors was validated by Western blotting (not shown). Confirming a role for AKT signaling in a TGF-b autocrine loop, formation of Nf1/ but not WT or TC21/;Nf1/ spheres was significantly reduced by shAKT (Fig. 4D). Thus, formation and survival of Nf1/spheres in vitro are dependent on TC21, TGF-b, TGFbRI, and AKT. We tested whether loss of TC21 affects signaling in vivo by immunochemistry. We stained E12.5 DRG tissue sections containing SCPs, the targets for Nf1 loss in the Nf1fl/fl;DhhCre model, with antiphospho-AKT. Nf1/ DRG SCPs showed elevated levels of phospho-AKT compared with WT and TC21/;Nf1/ embryos (Fig. 4E and F). Nf1/ spheres from E12.5 DRG grown in vitro also contained increased phosphoAKT compared with WT and TC21/;Nf1/ spheres (Supplementary Fig. S4B). P-AKT was similar in neurofibromas from Nf1fl/fl;DhhCre and Nf1fl/fl;DhhCre;TC21/ mice, consistent with TC21 affecting events shortly after Nf1 loss (not shown). Phospho-SMAD2/3 staining, marking canonical TGF-b signaling was similar across genotypes (Supplementary Fig. S4C and S4D). Thus, losing TC21 in an Nf1/ background

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Figure 3. A TGF-b autocrine loop in / SCPs. A, SCP spheres Nf1 treated with TGF-b1 and then counted (p ¼ 0.005 ANOVA). B, qRTPCR mRNA expression of TGF-b1 in spheres compared with WT spheres (t test: Nf1/ vs. TC21/;Nf1/). C, ELISA for TGF-b1 protein. D and E, phase contrast micrographs of Nf1/ spheres after treatment. F, quantification of Nf1/ spheres after treatment.

correlated with reduced expression of phospho-AKT in SCPs in vivo. To test if TGF-b1 mRNA production in SCPs requires AKT, we examined levels of TGF-b1 mRNA in Nf1/ SCPs treated with the AKT inhibitor; the inhibitor decreased TGF-b1 mRNA (Fig. 4G). These data support an existence of a TC21-dependent TGF-b autocrine survival loop, requiring AKT, in Nf1/ SCP cells (Fig. 4H). We propose that absence of the autocrine loop causes the observed delay in benign tumor formation and decreased survival of neurofibromabearing mice. Loss of TC21 in MPNST cells increases tumor growth In the NPC is model of GEM-sarcoma and GEM–glioblastoma multiforme, loss of TC21 caused early lethality (Fig. 1B). Brain tumors killed mice before the normal onset of sarcoma formation in the NPCis model. We showed that TC21 activity is high in human MPNST S462TY cells that are NF1/ in comparison with NF1 WT 26T cells, using a Ras pull-down assay and blotting with anti-TC21 antibody (Supplementary Fig. S5A). To investigate the role of TC21 in sarcomas, we infected the S462TY cells with a stably expressing doxycyclineinducible TRIPZ lentivirus encoding shRNA targeting TC21 (shTC21), or control TRIPZ nontarget shRNA (NT). shTC21 cells were injected subcutaneously into the right flank of nu/nu mice and NT cells into the left flank and tumor growth measured. Tumors with shTC21 cells were larger than tumors with NT cells (Fig. 5A and B). Western blot analysis confirmed low TC21 expression in excised tumors (Fig. 5C). Thus, loss of TC21 in a human sarcoma xenograft increases tumor growth. However, histologic analysis of shTC21 MPNST xenografts revealed no differences in morphology (H&E) or cell proliferation (Ki67) compared with controls (not shown). As TGF-b can induce blood vessel formation we analyzed tumor vasculature. The shTC21 tumors had more meca-32þ vessels/mm3 than controls (Fig. 5D). Consistent with altered tumor stroma upon TC21 loss, an antibody against smooth muscle actin


(SMA) detecting myofibroblasts revealed increased immunoreactivity in xenografts expressing shTC21 (Fig. 5E and F). Increased MPNST growth due to loss of TC21 requires TGF-b To investigate whether TGF-b plays a causal role in malignant tumor growth when TC21 is lost, we treated mice harboring MPNST xenografts with function-blocking antiTGF-b1, 2, 3 in vivo (35). After tumors grew to approximately 250 mm3, rabbit IgG or anti-TGF-b antibody was administered by intraperitoneal injection. Tumors expressing shTC21 were larger than those with NT (Fig. 6A). Strikingly, mice injected with the anti-TGF-b antibody had tumors of similar size whether the cells were expressing shTC21 or control (Fig. 6B). Western blot analysis of tumor xenografts expressing shTC21 confirmed sustained reduction of TC21 (not shown). Thus, loss of TC21 in NF1/ MPNST cells increases tumor growth and requires TGF-b. To delineate signaling pathways modulated by the TGF-b antibody, we examined expression of phospho-SMAD in tumor sections. Phospho-SMAD was reduced in tumor cells and stroma from mice treated with the anti-TGF-b antibody compared with those treated with IgG (Fig. 6D and E). We hypothesized that, as in neurofibromas, in the malignant setting with increased TGF-b expression we might detect increased phospho-AKT. Indeed, phospho-AKT was elevated in tumor cells expressing shTC21. Phospho-AKT levels were subsequently reduced upon treatment with anti-TGF-b antibody (Fig. 6C and E). The shTC21 tumors treated with anti-TGF-b also had significantly fewer meca-32þ vessels than controls (Supplementary Fig. S5B). Together this data suggests that in the NF1/ setting, in transformed cells, loss of TC21 drives tumorigenesis in a TGF-b—dependent manner; TGF-b produced by MPNST cells acts in a nonautonomous fashion on stromal cells to increase blood vessels and promotes myofibroblasts formation.



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Figure 4. Survival of Nf1 mutant SCPs is dependent on TGF-b and AKT. SCP spheres treated with anti-TGF-b antibody (A), TGFbR1 inhibitor (B), SB 431542, and AKT inhibitor (C), MK-2206, or shAKT (D) compared with NT. Spheres counted after treatment. E, E12.5 DRGs stained with antiphospho-AKT. Adjacent sections stained with antineurofilament (to highlight neurons in DRG). Scale bar, 50 mm. F, quantification of staining intensity for phospho-AKT / SCP spheres after treatment. H, model of TC21-dependent TGF-b autocrine loop in in DRG sections. G, qRT-PCR, TGF-b1 mRNA expression in Nf1 Nf1/ SCPs.

MPNST cells express TGF-b and lose TGFbRII We tested if TGF-b ligands or TGFbRs are altered in neurofibromas or MPNST. We measured relative abundance of TGF-b ligands and receptor mRNAs in a panel of human neurofibromas and MPNSTs compared with cultured neurofibroma Schwann cells, MPNST cell lines, normal human Schwann cells, and normal human nerves by Affymetrix gene expression analysis. The heat map (Fig. 7A) shows upregulation of TGF-b3 in most neurofibromas and MPNST, with TGF-b2 upregulation specifically in 50% of neurofibroma Schwann cell samples, 50% MPNST cell lines and all human MPNST. TGFbRII was downregulated in all human MPNSTs, and in a subset (22/48) of neurofibromas and in neurofibroma Schwann cell samples (Fig. 7A). TGFbRI mRNA was slightly but not significantly upregulated. These data are consistent with findings that TGF-b ligands increase and receptors decrease in many tumor types during progression to malignancy (16). To confirm increased expression of TGF-b in MPNST and test if TGF-b expression is regulated by TC21 in sarcoma cells (as in SCPs), we examined TGF-b mRNA and protein expressions in NF1 human MPNST cells and in sarcomas from NPCis mice. In

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benign neurofibromas and SCPs, loss of TC21 reduced TGF-b1 mRNA levels in the Nf1/ setting. In Nf1 mutant mouse NPCis tumors TGF-b1 was elevated on loss of TC21, but TGF-b2 mRNA was not (Supplementary Fig. S5C and S5D). In NF1/ human MPNST shTC21 xenotransplants, TGF-b2 mRNA levels were significantly elevated in tumors (Fig. 7B) whereas TGF-b1 and TGF-b3 levels were unchanged (Supplementary Fig. S5E and S5F). Thus in mouse and human, TGF-b levels are regulated by TC21, but the 2 species use different TGF-b ligands. We examined the concentration of TGF-b2 protein in the media of shTC21 or control S462TY cells. Knockdown of TC21 in the NF1/ MPNST cells caused a 80-fold increase in secreted TGF-b2 (Fig. 7C). The pathway leading to upregulation of TGF-b mRNA remains uncertain. Blockade of the MEK, AKT, p38, and ROCK kinases failed to alter TGF-b mRNA levels in MPNST cells (not shown). TP53 mutation status may be important, as no upregulation of TGF-b2 mRNA was identified in the single existing NF1 MPNST cell line known to have WT p53 (Supplementary Fig. S5G). TGFbRs are frequently mutated or lost in malignancy (15). If TGFbRs show loss of function in NF1, then TGF-b effects would

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Figure 5. Loss of TC21 in MPNST cells increases sarcoma growth. A, volume quantification of xenograft tumors over time. B, gross photographs of nude mice tumors. C, Western blot showing TC21 protein in S462TY xenograft tumors. D, quantification of anti-meca staining and measurement of numbers of vessels/high-power field. E and F, SMA immunostaining in xenograft sections (brown). Scale bar, 50 mm.

be unlikely to be cell autonomous. TGFbRII protein was undetectable by Western blotting in S462TY cells compared with an immortalized human Schwann cell line (Fig. 7D). In 2 of 3 additional human MPNST cell lines, TGFbRII protein was also reduced (Supplementary Fig. S5H). The absence of TGFbRII in S462TY cells predicted that the cells would not respond to TGF-b. Indeed, no effects of TGF-b on MPNST survival or migration were detected (not shown). To verify this finding, we examined expression of the TGF-b target genes, p21, p15, and c-myc (19). No significant change in mRNA expression of these genes was detected after addition of TGF-b to S462TY cells in serum-free medium at 30 minutes or 8 hours, in comparison with untreated cells (not shown). Thus, when TC21 is lost in NF1/ MPNST cells, TGF-b mRNA and secreted protein are increased (Fig. 7E). Because MPNST cells lack significant amounts of TGFbRII, TGF-b affects surrounding TGFbRII expressing endothelial and stromal fibroblasts, resulting in increased blood vessels, conversion of fibroblasts to myofibroblasts, and increased tumor growth.

Discussion Using Nf1 models predicted to activate Ras proteins expressed in neurofibromin-dependent cells enabled the dem-


onstration that the Ras-related protein TC21 critically regulates TGF-b in neurofibroma and MPNST. Loss of TC21 in the setting of Nf1 deficiency delayed formation of benign neurofibromas, accelerated formation of aggressive brain tumors and nerve sarcomas, and uniquely regulated expression of TGF-b. Our identification of a specific role for TC21 is consistent with recent studies showing differential localization of other Ras proteins (4), and different embryonic survival following knockout of the K-, H-, and N-Ras genes (5). Our in vivo studies showed that TC21 functions as an oncogene in benign tumors and revealed a new role for TC21 as a tumor suppressor in nervous system malignancy. However, TC21 was identified as upregulated in some cancers (36, 37) and activated in lymphoma in vivo (8). Therefore, effects of TC21 activation are likely to be cell-type dependent. Mechanistically, we showed that PI3K/AKT is a critical effector of TC21. In all NF1 models tested, TC21 regulated TGF-b, and TGF-b acted as an oncogene. Loss of TC21 in GEM neurofibroma extended mouse survival without affecting neurofibroma number or size, suggesting effects on early stages of tumor formation. Consistent with a critical role for TC21 early in neurofibroma formation, we identified an autocrine survival loop specific to Nf1/ SCPs, and dependent upon TGF-b, TGF-bR, and the PI3K/AKT



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Figure 6. TGF-b mediates aggressive growth of MPNST xenografts. Volume quantification of xenograft tumors over time. Mice injected IgG (A) or anti-TGF-b (B) antibody. Quantification of staining intensity phospho-SMAD2/3 (C) and phospho-AKT (D) in xenograft tumors. Grading defined in Fig. 4 legend. E, immunohistochemistry staining of xenograft tumor sections. I.P., intraperitoneal; scale bar, 25 mm.

pathway. We have not excluded additional effects on more mature Schwann cells. TGF-b1 can kill mature WT Schwann cells as well as more immature cells (38, 39), and as in Nf1/ SCPs, adult Nf1/ nerves contained increased levels of TGF-b1 mRNA that depended on TC21. We failed to detect elevated levels of TGF-b1 mRNA in neurofibromas, but levels of TGF-b1 mRNA in Schwann cells might have been obscured by expression in neurofibroma mast cells or other cells in the tumor microenvironment (40). Additional evidence supporting a role for TC21 early in neurofibroma formation comes from clonality assays. Numbers of Nf1/ primary sphere-forming cells, after acute inactivation at E12.5 using the DhhCre allele, were reduced by loss of TC21. Nf1/ primary sphere-forming cells were dependent on TGF-b they produced for their own survival. Secreted TGF-b is predicted to enhance numbers of developing Nf1/ stem/ progenitor cells and thus tumorigenic potential, leading to neurofibroma formation. Consistent with this idea, targeted loss of the TGFbRII in Schwann cells suppressed early Schwann

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cell death and proliferation (41). We conclude that TC21 controls TGF-b in vivo, reducing the growth and number of developing Schwann cells, and thereby acting as a brake on neurofibroma initiation/growth. Precursor cell effects on neurofibroma initiation and/or growth through TC21 and TGF-b were not sufficient to prevent tumorigenesis. Other Ras proteins and/or Nf1 functions are likely necessary for neurofibroma growth. The idea that Nf1 regulates other Ras proteins—even in peripheral nerve cells—is supported by experiments in which farnesyl transferase inhibitors predominantly blocking H-Ras, blocks Nf1 mutant Schwann cell proliferation (42), and a report that N-Ras plays a major role in MPNST cells (43). Our finding that loss of TC21 delayed Nf1/ embryonic lethality—but did not rescue embryos to birth—is also consistent with roles of multiple Ras proteins in development. How each Ras protein contributes to embryonic development and tumorigenesis downstream of NF1 loss of function remains to be determined. In addition, other Ras proteins may regulate

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Figure 7. MPNST cells express TGF-b and lose TGFbRII. A, heat map of TGF-b ligands and receptor that differ in human neurofibromas (NF; n ¼ 22), MPNSTs (n ¼ 6), cultured human primary neurofibroma Schwann cells (NFSC; n ¼ 22), and MPNST cell lines (MPNST cell; n ¼ 13), compared with normal human Schwann cells (NHSC; n ¼ 10) and normal human peripheral nerves (Nerve; n ¼ 3). Fold change ¼ 1.5 and t test P value < 0.05 with false discovery rate (Benjamini–Hochberg) correction. Scale bar, relative gene expression as colors. B, qRT-PCR, TGF-b2 mRNA expression. C, ELISA for TGF-b2 protein concentration. D, Western blot analysis of iHSC and S462TY cells. E, model showing effects of TC21 on TGF-b in MPNST cells.

TGF-b expression or signaling in non-NF1 tumor settings. The diverse regulation of TGF-b pathways in specific cell types may result in part because specific Ras proteins are cell-type specific (44). We found that loss of TC21 enhances tumorigenesis in malignancy. In the NPCis mouse model, loss of TC21 dramatically accelerated formation of brain tumors. In a xenograft model, NF1/ sarcoma cells showed accelerated tumor growth and increased levels of TGF-b when they expressed shTC21. Furthermore, inhibiting TGF-b blocked the elevated tumor growth caused by loss of TC21. The action of TGF-b as a growth promoter in malignancy correlated with increased TGF-b ligands in neurofibroma and MPNST and decreased expression of TGFbRII in human MPNST cell lines and tumors, as shown by gene expression and confirmed by protein analysis in cell lines. Genomic mutation or loss of one or more TGFbRs is common in many tumor types (15, 45). Despite absence of


TGFbRII in S462TY cells, blocking TGF-b led to decreased expression of phospho-SMAD. This may result from use of a mutant p53 SMAD complex (18) and/or more complex activation of SMAD downstream of activin receptors. One possibility is that stromal cells secrete activins or bone morphogenetic protein that indirectly alter SMAD phosphorylation in tumor cells (16). Our data are consistent with important noncell autonomous effects of MPNST cell produced TGF-b. This interpretation is consistent with increased blood vessels per area. SMA (myofibroblast) expression also increased upon downregulation of TC21, and both phenotypes were blocked by anti-TGF-b antibody. A noncell autonomous effect of TGF-b in malignancy has been documented previously in models of carcinoma, using genetic loss of TGFbRII (19). In summary, TC21 has a dual effect: in developing Nf1 progenitor cells this Ras-related protein promotes cell survival



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by promoting TGF-b production and formation of an autocrine survival loop, so that loss of TC21 results in decreased precursors and delayed tumor formation. TC21 activation plays a role in SCP viability and growth rather than in neurofibroma growth per se. In MPNST cells loss of TC21 dramatically increases TGF-b production, increasing vascularization and tumor growth. Our results linking TC21 to regulation of TGF-b production are likely to be relevant to cancer in general, as NF1 mutations are increasingly identified in sporadic cancers (46–48). Disclosure of Potential Conflicts of Interest

Writing, review, and/or revision of the manuscript: D.M. Patmore, P.C. Fulkerson, M.H. Collins, T.P. Cripe, N. Ratner Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.M. Patmore, J.J. Kordich Study supervision: D.M. Patmore, T.P. Cripe, N. Ratner Assisted with Xenograft experiments—injections, cell manipulation, etc.: D. Eaves

Acknowledgments The authors thank E. Ruoslahti (Sanford-Burnham Medical Research Institute) for R-Ras and TC21 mice, Luis Parada (UT Southwestern) for Nf1fl/fl mice, D. Meijer (Erasmus MC, Netherlands) for DhhCre mice, and M. Wallace (University of Florida) for immortalized human Schwann cells. The authors thank N. Nassar (CCHMC) for manuscript review, D. Largaespada (University of Minnesota), S. Kozma, and G. Thomas (University of Cincinnati) for extensive discussion and comments on the manuscript.

No potential conflicts of interest were disclosed.

Authors' Contributions Conception and design: D.M. Patmore, T.P. Cripe, N. Ratner Development of methodology: D.M. Patmore, T.P. Cripe, N. Ratner Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.M. Patmore, S. Welch, P.C. Fulkerson, J. Wu, N. Ratner Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.M. Patmore, S. Welch, P.C. Fulkerson, K. Choi, M.H. Collins, T.P. Cripe, N. Ratner

Grant Support This work was supported by NIH P50NS057531 to N. Ratner. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received May 21, 2012; revised July 19, 2012; accepted July 27, 2012; published OnlineFirst August 23, 2012.

References 1. 2. 3. 4.

5. 6.

7.

8.

9.

10. 11.

12.

13.

14.

15. 16.

5326

Mitin N, Rossman KL, Der CJ. Signaling interplay in Ras superfamily function. Curr Biol 2005;15:R563–74. Castellano E, Downward J. Role of RAS in the regulation of PI 3-kinase. Curr Top Microbiol Immunol 2010;346:143–69. Karnoub A, Weinberg R. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 2008;9:517–31. Quinlan M, Quatela S, Philips M, Settleman J. Activated Kras, but Not Hras or Nras, may initiate tumors of endodermal origin via stem cell expansion. Mol Cell Biol 2008;28:2659–74. Fernandez-Medarde A, Santos E. Ras in cancer and developmental diseases. Genes Cancer 2011;2:344–58. Graham S, Oldham S, Martin C, Drugan J, Zohn I, Campbell S, et al. TC21 and Ras share indistinguishable transforming and differentiating activities. Oncogene 1999;18:2107–16. Chan A, Miki T, Meyers K, Aaronson S. A human oncogene of the RAS superfamily unmasked by expression cDNA cloning. Proc Natl Acad Sci U S A 1994;91:7558–62. Kim R, Trubetskoy A, Suzuki T, Jenkins NA, Copeland NG, Lenz J. Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas. J Virol 2003;77: 2056–62. Ohba Y, Mochizuki N, Yamashita S, Chan A, Schrader J, Hattori S, et al. Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3. J Biol Chem 2000;275:20020–6. Boyd K, Korf B, Theos A. Neurofibromatosis type 1. J Am Acad Dermatol 2009;61:1–14. Evans D, Baser M, McGaughran J, Sharif S, Howard E, Moran A. Malignant peripheral nerve sheath tumours in neurofibromatosis type 1. J Med Genet 2002;39:311–4. Huang Y, Rangwala F, Fulkerson PC, Ling B, Reed E, Cox AD, et al. Role of TC21/R-Ras2 in enhanced migration of neurofibromin-deficient Schwann cells. Oncogene 2004;23:368–78. Erdogan M, Pozzi A, Bhowmick N, Moses HL, Zent R. Signaling pathways regulating TC21-induced tumorigenesis. J Biol Chem 2007;282:27713–20. s A, Me rida I, Delgado P, Cubelos B, Calleja E, Martínez-Martín N, Cipre et al. Essential function for the GTPase TC21 in homeostatic antigen receptor signaling. Nat Immunol. 2009;10:880–8. J. TGFb in cancer. Cell 2008;134:215–30. Massague Yang L, Pang Y, Moses HL. TGFb and immune cells: an important regulatory axis in the tumor microenvironment and progression. Trends Immunol 2010;31:220–7.


17. Rahimi R, Leof E. TGF-beta signaling: a tale of two responses. J Cell Biochem 2007;102:593–608. 18. Adorno M, Cordenonsi M, Montagner M, Dupont S, Wong C, Hann B, et al. A mutant-p53/Smad complex opposes p63 to empower TGFbinduced metastasis. Cell 2009;137:87–98. 19. Lu S, Herrington H, Reh D, Weber S, Bornstein S, Wang D, et al. Loss of transforming growth factor-beta type II receptor promotes metastatic head-and-neck squamous cell carcinoma. Genes Dev 2006;20: 1331–42. J. A mechanism of 20. Kretzschmar M, Doody J, Timokhina I, Massague repression of TGFb/Smad signaling by oncogenic Ras. Genes Dev 1999;13:804–16. 21. Lee M, Pardoux C, Hall M, Lee P, Warburton D, Qing J, et al. TGF-b activates Erk MAP kinase signalling through direct phosphorylation of ShcA. EMBO 2007;26:3957–67. 22. Grusch M, Petz M, Metzner T, Ozturk D, Schneller D, Mikulits W. The crosstalk of RAS with the TGF-b family during carcinoma progression and its implications for targeted cancer therapy. Curr Cancer Drug Targets 2010;10:849–57. 23. Geiser A, Kim S, Roberts A, Sporn M. Characterization of the mouse transforming growth factor-beta 1 promoter and activation by the Haras oncogene. Mol Cell Biol 1991;11:84–92. 24. Yue J, Mulder K. Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway. J Biol Chem 2000;275: 30765–73. 25. Cordenonsi M, Montagner M, Adorno M, Zacchigna L, Martello G, Mamidi A, et al. Integration of TGF-beta and Ras/MAPK signaling through p53 phosphorylation. Science 2007;315:840–3. 26. Wu J, Williams J, Rizvi T, Kordich J, Witte D, Meijer D, et al. Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert Hedgehog-expressing cells. Cancer Cell 2008;13:105–16. 27. Brannan C, Perkins A, Vogel K, Ratner N, Nordlund M, Reid S, et al. Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev 1994;8:1019–29. 28. Reilly K, Loisel D, Bronson R, McLaughlin M, Jacks T. NF1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nat Genet 2000;26:109–13. 29. Williams J, Wu J, Johansson G, Rizvi T, Miller S, Geiger H, et al. Nf1 mutation expands an EGFR-dependent peripheral nerve progenitor

Cancer Research




30.

31.

32.

33.

34.

35.

36.

37.

38.

that confers neurofibroma tumorigenic potential. Cell Stem Cell 2008;3:658–69. Ghadimi M, Young E, Belousov R, Zhang Y, Lopez G, Lusby K, et al. Survivin is a viable target for the treatment of malignant peripheral nerve sheath tumors. Clinical Cancer Res 2012;18:2545–57. Frahm S, Mautner V, Brems H, Legius E, Debiec-Rychter M, Friedrich R, et al. Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve sheath tumors of neurofibromatosis type 1 patients. Neurobiol Dis 2004;16:85–91. Miller SJ, Rangwala F, Williams JP, Ackerman P, Kong S, Jegga A, et al. Large-scale molecular comparison of human Schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues. Cancer Res 2006;66:2584–91. Miller SJ, Jessen WJ, Mehta T, Hardiman A, Sites E, Kaiser S, et al. Integrative genomic analyses of neurofibromatosis tumours identify SOX9 as a biomarker and survival gene. EMBO Mol Med 2009;1: 236–48. Stemmer-Rachamimov A, Louis D, Nielsen G, Antonescu C, Borowsky A, Bronson R, et al. Comparative pathology of nerve sheath tumors in mouse models and humans. Cancer Res 2004;64: 3718–24. Habashi JP, Judge DP, Holm TM, Cohn RD, Loeys BL, Cooper TK, et. al. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science 2006;312:117–21. Arora S, Matta A, Shukla N, Deo S, Ralhan R. Identification of differentially expressed genes in oral squamous cell carcinoma. Mol Carcinog 2005;42:97–108. Sharma R, Sud N, Chattopadhyay T, Ralhan R. TC21/R-Ras2 upregulation in esophageal tumorigenesis: potential diagnostic implications. Oncology 2005;69:10–8. Hagedorn L, Suter U, Sommer L. P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors. Development 1999;126: 3781–94.


39. Parkinson D, Dong Z, Bunting H, Whitfield J, Meier C, Marie H, et al. Transforming growth factor b (TGFb) mediates Schwann cell death in vitro and in vivo: examination of c-Jun activation, interactions with survival signals, and the relationship of TGFb-mediated death to Schwann cell differentiation. J Neurosci 2001;21: 8572–85. 40. Yang FC, Chen S, Clegg T, Li X, Morgan T, Estwick SA, et al. Nf1þ/ mast cells induce neurofibroma like phenotypes through secreted TGF-signaling. Hum Mol Genet 2006;15:2421–37. 41. D'Antonio M, Droggiti A, Feltri ML, Roes J, Wrabetz L, Mirsky R, et al. TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves. J Neurosci. 2006; 26:8417–27. 42. Kim H, DeClue J, Ratner N. cAMP-dependent protein kinase A is required for Schwann cell growth: interactions between the cAMP and neuregulin/tyrosine kinase pathways. J Neurosci Res 1997;49: 236–47. 43. Kraniak J, Sun D, Mattingly R, Reiners JJ, Tainsky M. The role of neurofibromin in N-Ras mediated AP-1 regulation in malignant peripheral nerve sheath tumors. Mol Cell Biochem 2010;344:267–76. 44. Rahimi R, Leof E. TGFbeta versatility: PI3K as a critical mediator of distinct cell type and context specific responses. Cell Cycle 2009;8:1813–7. 45. Akhurst R, Derynck R. TGF-beta signaling in cancer-a double-edged sword. Trends Cell Biol 2001;11:S44–51. 46. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008;455:1069–75. 47. Network CGA. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008;455: 1061–8. € lzel M, Huang S, Koster J, ra I, Lakeman A, Caron H, et al. NF1 is a 48. Ho tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell 2010;142:218–29.



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In Vivo Regulation of TGF-β by R-Ras2 Revealed through Loss of the RasGAP Protein NF1 Deanna M. Patmore, Sara Welch, Patricia C. Fulkerson, et al. Cancer Res 2012;72:5317-5327. Published OnlineFirst August 23, 2012.

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