Induction of Nonapoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

Published OnlineFirst August 16, 2010; DOI: 10.1158/1541-7786.MCR-10-0090 Published OnlineFirst on September 21, 2010

Cell Cycle, Cell Death, and Senescence Molecular Cancer Research Induction of Nonapoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

Haymanti Bhanot, Ashley M. Young, Jean H. Overmeyer, and William A. Maltese

Abstract Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin- independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1 (G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death. Mol Cancer Res; 8(10); OF1–17. ©2010 AACR.

Introduction tion of Ras signaling pathways. We recently determined that this form of cell death is morphologically and mecha- The reduced sensitivity of many types of cancer cells to nistically distinct from the various types of cell death noted apoptotic cell death has stimulated interest in identifying above (15). It entails a stimulation of macropinocytosis (cell alternative nonapoptotic cell death pathways that might drinking), combined with defects in clathrin-independent be used to kill tumor cells in a therapeutic context. Several endocytic vesicle trafficking, ultimately resulting in cellular different forms of nonapoptotic cell death have been disruption as large vacuoles expand to fill the cytoplasmic described based on specific cellular or molecular criteria. space. We have termed this form of cell death “methuosis” These include autophagy-associated cell death (1-3), para- from the Greek methuo (to drink to intoxication). Morpho- ptosis (4, 5), oncosis (6-8), necroptosis (9, 10), entosis logically, methuosis resembles type IIIA nonlysosomal (11), and programmed necrosis (12, 13). necrotic cell death (16), originally observed in vacuolated A decade ago, Chi et al. (14) reported a unique form of cartilage during mineralization (17). It also bears some nonapoptotic cell death that could be induced in glioblas- similarity to the extensive endosomal vacuolization noted toma and gastric carcinoma cells by constitutive stimula- in gastric epithelial cells exposed to the Helicobacter pylori VacA cytotoxin (18, 19) and in regions of liver necrosis induced by furosemide (20). Despite these superficial sim- Authors' Affiliation: Department of Biochemistry and Cancer Biology, ilarities, it remains unclear whether these forms of cell death University of Toledo College of Medicine, Toledo, Ohio share a common molecular mechanism. Although it is not Note: Supplementary data for this article are available at Molecular yet known if methuosis occurs in normal physiologic con- Cancer Research Online (http://mcr.aacrjournals.org/). texts, it is possible that understanding the molecular signals Corresponding Author: William A. Maltese, Department of Biochemistry that drive this nonconventional form of cell death may and Cancer Biology, Block Health Sciences Building, Mail Stop 1010, University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, prove useful for devising new approaches to eliminate can- OH 43614. Phone: 419-383-4161; Fax: 419-383-6228. E-mail: william. cer cells that are refractory to apoptosis. [email protected] We have found that stimulation of vacuolization in glio- doi: 10.1158/1541-7786.MCR-10-0090 blastoma cells by activated forms of H- and K-Ras does not ©2010 American Association for Cancer Research. depend on conventional Ras effector pathways such as the

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Raf-MEK-ERK kinase cascade or the phosphatidylinosi- Tight-Pur (Clontech). Retrovirus was produced by trans- tide 3′-kinase (PI3K)–Akt pathway (21). In considering fecting these constructs together with pRetroX-VSVG- alternative mechanisms, we noted that previous studies Env into 293 GP2 packaging cells (Clontech) using had implicated the Rac1 GTPase as a positive regulator LipofectAMINE Plus reagent. Cells were maintained in of macropinocytosis (22), phagocytosis (23), and cellular DMEM + 10% heat-inactivated FBS, using Biosafety Level vacuolization that occurs in response to the VacA cytotoxin 2 precautions. Virus-enriched medium was passed through a (18). Because downstream targets of Ras include guanine 0.45-μm filter and used to infect the U251Tet-On cells. nucleotide exchange factors (GEF) that can stimulate acti- Cells were maintained in medium containing 1 μg/mL of vation of Rac1 (24), we hypothesized that Ras might work puromycin and 200 μg/mL of G418. For the expression through Rac to trigger methuosis. Consistent with this of the genes of interest, 1 μg/mL of doxycycline was added possibility, we observed that expression of constitutively to the medium. active Rac1 (15), but not the related GTPases, Cdc42 or RhoA (21), could mimic the effects of Ras in glioblastoma Nucleofection and retroviral expression of proteins cells. Based on these findings, we initiated the present Myc-tagged H-Ras(G12V) and H-Ras(G12V, C186S) study to test the hypothesis that Rac1 is an essential down- cDNAs were generated and cloned into the pCMV5 stream mediator of Ras-induced methuosis. The results expression vector as described previously (21). pCMV5- clarify the pathway for Ras-induced methuosis by estab- myc-Rac1(G12V) was provided by A.L. Wilson-Delfosse lishing that the underlying perturbations in macropino- (Case Western Reserve University, Cleveland, OH). some/endosome trafficking that lead to cell vacuolization U251 cells were nucleofected with an Amaxa Nucleofector require both activation of Rac1 and a coordinate Rac1- II system, using Solution T and Program T30, according to dependent decrease in the pool of active Arf6, a GTPase the manufacturer's protocol (Lonza). For each reaction, implicated in endocytic trafficking. The latter is mediated 5 μg of plasmid DNA was introduced into 3 × 106 cells through an interaction between the active form of Rac1 that had been harvested in RPMI 1640 supplemented with and an Arf GAP, GIT1. 10% heat-inactivated FBS. Following nucleofection, cells were plated and analyzed 18 to 20 hours later. Materials and Methods For constitutive expression of myc-Rac1(G12V) or myc- H-Ras(G12V) in various cell lines, the cDNAs encoding Cell lines for conditional expression of Ras and the latter constructs were cloned into the pQCXI-H retro- Rac1 constructs viral vector (Clontech) using the NotI and BamHI sites. Ret- U251 human glioblastoma cells were purchased from rovirus was produced in 293 GP2 packaging cells (Clontech) the DCT Tumor Repository (National Cancer Institute). cotransfected with pQCXI-H-Rac1(G12V) and pRetroX- U20S human osteosarcoma cells were obtained from VSVG-Env (Clontech), using LipofectAMINE Plus reagent. American Type Culture Collection. HEK293T cells were At 48 hours after transfection, the virus-enriched medium obtained from Systems Biosciences. Unless stated other- was collected, filtered, and used to infect the cells. After wise, cell lines were grown in DMEM supplemented with 24 hours, the virus was removed and the cells were main- μ 10% fetal bovine serum (FBS), at 37°C with 5% CO2. tained in DMEM with 10% FBS, 1 g/mL puromycin, Generation of the U251-C18 cell line, which expresses and 200 μg/mL of hygromycin until they were examined. myc-H-Ras(G12V) under the control of a tetracycline (tet)-inducible promotor, was described previously (15). Short hairpin RNA–mediated gene silencing The U251-C18 cells were maintained in DMEM sup- For silencing expression of Tiam1, oligonucleotides plemented with 10% tetracycline-free FBS (Clontech), corresponding to nucleotides 5263 to 5283 [short hairpin 200 μg/mL of G418 and 200 μg/mL of hygromycin. RNA (shRNA) no. 1, 5′-CCGTAGAGAATGTGTGTA- A stable U251 cell line that expresses myc-Rac1(G12V) GAT-3′] in the 3′ untranslated region of huTiam1 (acces- under the control of a tetracycline-inducible promoter, sion number U16296) or 3157 to 3177 of the coding ′ herein designated as U251-Rac1(G12V)tet, was generated region (shRNA no. 2, 5 -CGCACCTACGTGAAGGATT- as described previously (15). TA-3′) were used. Each oligonucleotide was followed by a To generate new stable cell lines that conditionally ex- 12-base loop region (CTTCCTGTCAGA) and the reverse press activated Ras or dominant negative Rac1 or both, complement of the 21-bp gene-specific sequence. The U251 cells were infected with virus-enriched medium hairpin sequences were flanked by BamHI and EcoRI containing the regulatory vector pRetroX-Tet-On (Clon- restriction sites. The oligonucleotides were phosphorylated tech), which encodes the reverse tetracycline-regulated and annealed to their respective reverse complementary transactivator. Cells were selected in medium containing sequences, then ligated into the pSIH1-H1-Puro lentivec- 200 μg/mL of G418, and clonal cell lines were tested in tor (System Biosciences). transient transfection assays to determine which ones gave shRNA constructs to suppress expression of Eps8 or the tightest doxycycline-regulated gene expression. These GIT1 were made as described above except with different were designated as U251 Tet-On. cDNAs encoding myc- oligonucleotides: for Eps8, bases 1543 to 1563 (5′-CCA- H-Ras(G12V) and FLAG-Rac1(T17N) were subcloned ACTTCTAATCGCCATATA-3′) of huEps8 (accession into the NotI/MluI sites of the expression vector, pRetroX- number NM_004447). For GIT-1, bases 816 to 834

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(5′-GCACACCCATTGACTATGC-3′) and 1070 to 1090 Fluor 568 (Invitrogen). Cells were examined with a Nikon (5′-GAGGTGGATCGAAGAGAAAAT-3′) of huGIT1 Eclipse TE2000-U fluorescence microscope equipped with (accession number NM_014030; ref. 25). Control shRNA a digital camera and NIS-Elements AR software (Nikon constructs used oligonucleotides targeting Chiridius poppei Instruments, Inc.), and the percentage of vacuolated cells GFP (5′-GGAGGAGCTGCACAGCAA-3′)orAequorea was determined by counting at least 70 cells in photomi- victoria GFP (5′-GCAAGCTGACCCTGAAGTTC-3′), crographs from randomly selected fields. Criteria for neither of which match sequences in the human genome. counting cells as vacuolated were the same as those noted shRNA lentivirus was produced by simultaneous trans- above for phase contrast micrographs. fection of 293TN cells with shRNA-pSIH1-H1-Puro vector, pPackH1-Rev, pPackH1-Gag, and pPackVSV-G Uptake of fluid phase tracers (System Biosciences), using LipofectAMINE Plus reagent Endocytic compartments were labeled with the fluid phase (Invitrogen). Virus collected between 24 and 48 hours af- tracer, Lucifer yellow, as described previously (15). For label- μ ter transfection was passed through a 0.45- mfilterand ing cells with dextran-Alexa Fluor 568 (AF568), U251 C-18 used to infect U251 C-18 cells that were ∼50% confluent. cells were grown in 60 mm dishes for 3 days in phenol red– Cells were exposed to the virus for 2 to 3 days. One day free medium in the presence or absence of 1.0 μg/mL of after removal of the virus, doxycycline was added to the doxycycline and 20 μmol/L of EHT 1864, with medium re- medium (1 μg/mL) to induce myc-H-Ras(G12V) expres- placement each day. To quantify dextran uptake, cells were sion, and cells were analyzed 2 to 3 days later by phase incubated with 0.5 mg/mL of dextran-AF568 (Invitrogen) contrast microscopy or Western blotting. for 15 minutes, washed with HBSS, and harvested by trypsi- nization. Cells were suspended in a final volume of 500 μL, Cell morphology and viability and 100 μL of cell suspension was transferred to a black- To determine the percentage of cells with cytoplasmic walled, black-bottomed microplate and analyzed on a Spec- vacuoles, phase contrast images of live cells were captured traMax M5 plate reader (Molecular Devices) with 568 nm with an Olympus IX70 microscope equipped with a digital excitation and 603 nm emission filters. Background fluores- camera and SPOT imaging software (Diagnostics Ins- cence was subtracted using values obtained from cells incu- truments, Inc.). For each culture, multiple photographs bated in the absence of dextran-AF568. were taken of different fields of cells and at least 100 cells were scored for vacuolization. Cells containing three or Regulated ras expression with the ProteoTuner system more phase-lucent vacuoles with diameters ≥0.5 μm The Lenti-X ProteoTuner System was purchased from or with >10 smaller vacuoles (0.1-0.5 μm) were counted Clontech. Myc-tagged H-Ras cDNA constructs (G12V or as positive. G12V, C186S) were subcloned into the EcoRI-BamHI re- Cell viability was determined using the 3-(4,5- striction sites of the lentiviral expression vector, pLVX- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide PTuner Green, placing the Ras constructs in frame with assay, as described previously (26). PARP cleavage in the the sequence encoding the 12 kDa destabilization domain presence or absence of zVAD-fmk (Bachem Biosciences) (DD) and upstream from an IRES, which allows constitu- was determined by Western blot analysis. tive expression of ZsGreen1. Lentivirus was produced in Where replicate assays were done, statistical significance HEK293T cells (American Type Culture Collection) using of changes in cell morphology or viability was assessed by LipofectAMINE Plus reagents (Invitrogen), and pPack-H1 Student's t test. Lentiviral Packaging System (System Biosciences). Forty- eight hours after transfection, the medium containing the Western blot analysis virus was collected and filtered through a 0.45-μm mem- The 9E10 monoclonal antibody against the myc epitope brane. U251 glioblastoma cells were infected with the fil- (Calbiochem/EMD Biosciences) and the antibody to tered virus to express DD-myc-H-Ras(G12V). Four days GIT1 (BD Biosciences) were purchased from the indicated after infection, Shield1 was added to the medium at concen- sources. Antibody against α-tubulin was obtained from trations of 30 or 100 nmol/L to stabilize the DD tag and Sigma. The antibody against PARP was obtained from allow varying levels of Ras expression. Twenty-four hours af- BD Biosciences. Methods for protein determination, ter the addition of Shield1, overall infection efficiency in all SDS-PAGE, and Western blot analysis have been described cultures was determined to be >85% by counting the per- (27). Chemiluminescence signals were quantified using an centage of green fluorescent cells. The amount of DD-myc- Alpha Innotech FluorChem HD2 imaging system. H-Ras expressed was determined by immunoblotting with anti-myc monoclonal antibody or anti-Ras antibody M-90 Immunofluorescence (Santa Cruz Biotechnology). Cell morphology was assessed Cells were prepared for immunofluorescence as de- by phase contrast microscopy. The relative amount of acti- scribed previously (28). Myc-tagged proteins were detected vated Rac1 was assayed in cell lysates as described below. with a rabbit polyclonal antibody (EMD Biosciences) conjugated to FITC. FLAG-tagged proteins were detected Rac1 and Arf6 activation assays with a mouse monoclonal antibody, M2, from Sigma, Rac1 activation assays were done using the EZ-Detect followed by goat anti-mouse IgG conjugated with Alexa Rac1 Activation Kit (Thermo Scientific Pierce). The assay

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uses a GST-fusion protein containing the p21-binding observed with H-Ras(G12V) (15). This is not a general ef- domain of p21-activated protein kinase 1 (PAK1) to selec- fect of all Ras-related GTPases, as active Cdc42(G12V) tively bind active Rac1 in whole cell lysates. The active and RhoA(G14V) did not cause vacuolization (21). The Rac1 is then collected on glutathione agarose beads and accumulation of vacuoles induced by Rac1(G12V) repre- quantified by immunoblot analysis using anti-Rac1 mono- sents the first observable step in a pathway that ultimately clonal antibody (Thermo Scientific Pierce). The value for leads to cell detachment from the substratum and disrup- active Rac1 collected on the beads was normalized to the tion of the cell membrane. However, in these earlier stud- concentration of protein in the cell lysate. On a separate ies, we did not establish if the loss of cell viability caused immunoblot, the total Rac1 in a sample of the cell lysate by Rac1(G12V) was caspase-independent, as was the case was determined and normalized to the protein concentra- for H-Ras(G12V) (15). To examine this possibility, we in- tion. The normalized results were then expressed as the troduced myc-Rac1(G12V) into the U251 glioblastoma ratio of active Rac1 to total Rac1 in the sample. The same cell line using a retroviral vector. Within 48 hours, the approach was used to quantify active Arf6, except that the expression of Rac1(G12V) caused extreme cytoplasmic assay used GST-GGA3 (Golgi-associated gamma adaptin vacuolization (Fig. 1A), and within 5 to 7 days, there ear–containing Arf binding protein 3) to pull down active was a loss of cells from the substratum and a decrease in Arf6, and a monoclonal antibody against Arf6 for subse- the number of viable cells compared with cells infected quent immunoblot analysis (Thermo Scientific Pierce). with the empty viral vector (Fig. 1D). Although caspase Where replicate assays were done, statistical significance activation could be detected by examining PARP cleavage of changes in activation of Rac1 or Arf6 was assessed by in the detached cells (Fig. 1C), addition of the general Student's t test. caspase inhibitor, zVAD-fmk, at a concentration that com- For analysis of Rac1 or Arf6 in stable cell lines, activation pletely blocked PARP cleavage did not protect the cells assays were done on cells harvested on the 3rd day after the from Rac1(G12V)-induced vacuolization and cell death addition of doxycycline to induce expression of H-Ras (Fig. 1A, C, and D). In this respect, the effects of Rac1 (G12V). Control cells were maintained without doxycy- (G12V) were essentially identical to the cell death pheno- cline. Where indicated, EHT 1864 (provided by ExonHit type induced by activated Ras, which we have termed Therapeutics, Paris, France) was added to the culture methuosis (15). Identical results were obtained when the medium at a concentration of 25 μmol/L to block Rac1 same experiment was done with a stable U251 cell line activation. Both doxycycline and EHT 1864 were replaced in which myc-Rac1(G12V) was conditionally expressed each day after treatment until the assays were done. For cell in response to doxycycline (Supplementary Fig. S1). cultures transiently nucleofected with H-Ras constructs, the Rac1 or Arf6 activation assays were done 18 to Induction of methuosis by activated H-Ras depends on 24 hours after nucleofection. activation of endogenous Rac The preceding observations, coupled with reports that Immunoprecipitation Ras can activate Rac1, suggested that Rac lies downstream For immunoprecipitation of myc-Rac(G12V), cells were from Ras in the signaling pathway for methuosis. To begin washed with HBSS and homogenized in immunoprecipi- testing this possibility, we asked if overexpression of consti- tation buffer: 10 mmol/L of Tris-HCl (pH 7.4), 1 mmol/L tutively active H-Ras(G12V) could stimulate the activation of MgCl2, 150 mmol/L of NaCl, 0.75% v/v NP40, with of endogenous Rac in U251 glioblastoma cells. Cells Complete Mini Protease Inhibitors (Roche). Lysates were were nucleofected with an expression vector encoding cleared by centrifugation at 10,000 × g for 30 minutes either myc-H-Ras(G12V) or the farnesylation-incompetent at 4°C, and 1/10th of the supernatant solution was re- myc-H-Ras(G12V, C186S), which is relatively ineffective served for checking the expression of myc-Rac1(G12V) at inducing vacuolization (ref. 21 and Fig. 2A and B). and endogenous GIT1. The remainder of the solution The pool of activated endogenous Rac GTPase was then was incubated with rabbit anti-myc agarose beads (Sigma) measured by an established assay that takes advantage of for 2 hours at 4°C. The beads were washed three times the selective interaction of Rac-GTP with the p21 binding with immunoprecipitation buffer and the bound proteins domain of the Rac effector, PAK1 (Fig. 2C and D). The were subjected to SDS-PAGE and immunoblot analysis for results of this experiment established that in cells overex- myc-Rac1(G12V) or GIT1, using monoclonal antibodies pressing H-Ras(G12V), vacuolization was accompanied noted above. by a robust activation of endogenous Rac. Neither vacuolization nor activation of endogenous Rac occurred in cells Results expressing H-Ras(G12V) that contained a COOH-terminal cysteine substitution that prevents farnesylation and Constitutively active Rac1 mimics the effect of active membrane association of the protein. Activation of Rac2 H-Ras in stimulating nonapoptotic cell death was not examined in glioblastoma cells because Rac2 is ex- Our previous studies showed that overexpression of pressed mainly in cells of hematopoietic lineage (29). Rac3 constitutively active Rac1(G12V) in glioblastoma cells is strongly expressed in neuronal tracts of the brain (30) and causes a striking accumulation of vacuoles that incorporate in neuronal cell lines (31), but we were unable to detect the fluid-phase marker, Lucifer yellow, similar to what we Rac3 expression in glioblastoma cells, using an antibody

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FIGURE 1. Expression of activated Rac1 induces caspase-independent death in U251 glioblastoma cells. A, cells were infected with retrovirus encoding myc-Rac1(G12V) or virus without a cDNA insert (empty vector, EV). On the day after infection, 50 μmol/L of z-VAD-fmk was added to half of the cultures, and the others were maintained without the caspase inhibitor. On day 6 after infection (A) live cells were examined by phase contrast microscopy, (B) Western blot analysis was done to verify expression of myc-Rac1(G12V), and (C) attached and detached cells were harvested for analysis of PARP cleavage. There were no detached cells in the EV controls. D, cells expressing Rac1(G12V) were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assays over a period of 7 d to compare their viability with and without zVAD (the inhibitor was replenished every day). The results shown in the bar graph are from three independent experiments (mean ± SD). The decreases in viable cells at day 5 and day 7 (±zVAD) were significant at P ≤ 0.04. specific for Rac3 (ref. 32; data not shown). Therefore, for all in the expression level of myc-H-Ras(G12V) in parallel further studies, we inferred that Rac1 was the predominant cultures (Fig. 3A). Under these conditions, the extent of form of Rac being measured in glioblastoma cells. cellular vacuolization (Fig. 3C and D) correlated with the Numerous studies have documented the ability of ac- amount of Rac1 activation (Fig. 3B). tivated Ras to function as an oncogene, promoting cell To verify a direct link between Ras-induced activation proliferation and cell survival through activation of the of Rac1 and the extensive cytoplasmic vacuolization that Raf-MEK-ERK and PI3K-Akt signaling pathways (33, precedes methuosis, we used the compound EHT 1864, 34). Our earlier results indicated that Ras-induced cyto- which has been established as a selective inhibitor of Rac plasmic vacuolization does not require the activation of GTPases (35). The compound acts by displacing bound Raf or PI3K signaling pathways (21). This suggests that nucleotide, thereby preventing interaction of Rac with its the induction of methuosis by H-Ras(G12V) might occur downstream effectors (35). The inhibitor studies were only when the level of activated Ras reaches a threshold donewithastableglioblastomacellline,U251-C18 sufficient to stimulate Rac1 activation. To examine this (15), in which conditional expression of myc-H-Ras possibility, we used the ProteoTuner system to express (G12V) is obtained in response to the addition of doxy- myc-H-Ras(G12V) as a fusion protein with an NH2- cycline (Fig. 4A). In the absence of the Rac inhibitor, terminal destabilizing domain that promotes rapid protea- doxycycline-induced expression of H-Ras(G12V) was somal degradation. By adding increasing amounts of the accompanied by a significant increase in the activation small molecule, Shield1, which binds to the DD and im- of endogenous Rac1 (Fig. 4B), and this coincided with pairs degradation, we were able to obtain graded increases extensive vacuolization (Fig. 4C and D). When the Rac

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FIGURE 2. Expression of activated H-Ras causes an increase in the amount of active Rac1 in U251 glioblastoma cells. A, cells were nucleofected with expression vectors encoding the indicated Ras constructs. After 24 h, live cells were examined by phase contrast microscopy and harvested for Western blot analysis to check the expression of myc-tagged Ras proteins. B, cells were scored for vacuolization as described in Materials and Methods. C, pull-down assays for activated Rac1 were done on lysates from two pooled 10-cm dishes of cells. Bottom blots, active endogenous Rac1 that bound to the GST-PAK1. Top blots, total Rac1 in an aliquot of the cell lysate. D, the immunoblot signals for active and total Rac1 were quantified and the units for active Rac1 were normalized to total Rac as described in Materials and Methods.

inhibitor, EHT 1864, was added together with doxy- H-Ras markedly suppressed vacuolization compared with cycline, there was no diminution of myc-H-Ras(G12V) cells expressing H-Ras(G12V) alone. To account for varia- expression (Fig. 4A), but the Ras-dependent increase in tions in transfection efficiency, immunofluorescence active Rac1 was blocked (Fig. 4B). Most notably, inhibit- microscopy was done to identify only those cells that were ing the activation of Rac1 strongly suppressed Ras-induced expressing myc-H-Ras(G12V), FLAG-Rac1(T17N), or the vacuolization (Fig. 4C and D) and significantly reduced combination of both constructs (Fig. 5B). The percentage cellular uptake of the fluid phase marker, Dextran-AF568 of vacuolated cells was then calculated using only the (Fig. 4E). labeled cells. The results confirmed the qualitative impres- To obtain an independent line of evidence that Rac1 sions of cellular vacuolization based on phase contrast functions downstream from H-Ras for initiation of microscopy of the live cells. Taken together with the pre- methuosis, we attempted to stably suppress the expression ceding studies with the Rac1 inhibitor, these observations of endogenous Rac1 with shRNA. However, we found that support the idea that activation of a Rac1-mediated path- loss of Rac1 adversely affected cell attachment and pro- way is necessary for the early perturbations of endocytic liferation. As an alternative, we took advantage of the trafficking that lead to cellular vacuolization associated well-known ability of the dominant-negative Rac1 with methuosis. (T17N) to interfere with the function of endogenous Rac1 (22, 36). For these studies, we used the pRetroX- Stimulation of Rac1 activation by H-Ras(G12V) does Tight-Puro vector, in conjunction with the RetroX- not depend on conventional exchange factors in Tet-On system to generate U251 cell lines capable of glioblastoma cells conditional expression of myc-H-Ras(G12V) alone, We next turned our attention to the question of how FLAG-Rac1(T17N) alone, or myc-H-Ras(G12V) + H-Ras(G12V) might promote the activation of Rac1 in FLAG-Rac1(T17N). As shown in Fig. 5A, within 48 hours the glioblastoma cells. A number of Rac GEFs can be of adding doxycycline to the medium, phase contrast mi- activated by PI(3,4,5)P3, the product of PI3K (37). Thus, croscopy revealed that the cells expressing H-Ras(G12V) one possibility is that Ras(G12V) stimulates Rac1 activa- were extensively vacuolated and had begun to detach from tion indirectly through positive modulation of PI3K. We the dish, whereas the cells expressing Rac1(T17N) showed believe that this is unlikely because our previous studies a minimal level of vacuolization, similar to the cells incu- have shown that overexpression of PTEN in U251 cells bated without doxycycline. Most notably, coexpressing (where PTEN is mutated) suppresses PI3K signaling but the dominant-negative Rac1 construct together with active does not prevent Ras-induced vacuolization (21). A more

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likely possibility is that at high expression levels, H-Ras Ras-induced Rac1 activation and cellular vacuolization, (G12V) can directly stimulate the activity of a Rac GEF. we used shRNA directed against Eps8 to dramatically Among the members of the Rac GEF family, Tiam1 stands reduce the level of the protein in U251-C18 cells prior out as a candidate because it contains a Ras-binding to inducing the expression of H-Ras(G12V) (Fig. 6A). domain and has been reported to promote the activation Despite the near complete suppression of Eps8 expression, of Rac1 in a Ras-dependent manner (24). the morphologic response of knockdown cells was similar To explore the possible contribution of Tiam1 to Ras- to that observed in control cells with normal levels of Eps8 induced methuosis, we used a lentiviral vector to introduce (Fig. 6B). anti-Tiam1 shRNA into the U251-C18 stable cell line. Taken together, the preceding results indicate that the two The shRNA suppressed Tiam1 to undetectable levels most obvious Ras-responsive Rac GEFs, Tiam1 and Sos1/ (Fig.6A),butdidnotpreventdoxycycline-inducible Eps8/E3b1, are not essential for the initiation of vacuoliza- expression of myc-H-Ras(G12V) (Fig. 6B). Under these tion in glioblastoma cells expressing H-Ras(G12V). conditions, expression of H-Ras(G12V) continued to trigger the vacuolar phenotype that is characteristic of methuo- Ras-induced activation of Rac1 causes a decrease in the sis, despite the suppression of Tiam1 (Fig. 6B). amount of active Arf6 Sos1 is another GEF that has the potential to respond to As we considered possible molecular mechanisms where- H-Ras(G12V) by activating Rac1. Although Sos1 is best by overexpression of H-Ras(G12V) and consequent acti- known for its ability to form a complex with the adapter, vation of endogenous Rac1 might cause dysfunction of Grb2, to facilitate activation of Ras in response to receptor endocytic compartments, our attention was drawn to a tyrosine kinase signals (38), it can also behave as a Rac series of studies that have defined functional connections GEF when assembled into a tricomplex with Eps8 and a between Rac1 and another small GTPase, Arf6. In its scaffolding protein, E3b1 (Abi-1) (39, 40). To determine active state, Arf6 promotes membrane recycling from if such a complex might be responsible for mediating clathrin-independent endosomes (CIE) to the plasma

FIGURE 3. Rac1 activation and cellular vacuolization correlate with increasing levels of H-Ras(G12V) expression in U251 glioblastoma cells. A, cells were grown in the presence of increasing concentrations of Shield1 to obtain graded expression levels of DD-myc-H-Ras(G12V) using the ProteoTuner system. The blot shows the expressed Ras protein detected with the myc antibody. In a separate blot (data not shown) an antibody against H-Ras was used to determine the ratio of expressed DD-myc-H-Ras(G12V) to endogenous H-Ras. The results were 1.4 with no Shield1, 3.0 with 30 nmol/L of Shield1, and 4.5 with 100 nmol/L of Shield1. B, 1 d after the addition of Shield1, the amount of active endogenous Rac1, normalized to total Rac1, was determined as described in Materials and Methods. C and D, cell morphology was assessed by phase contrast microscopy and the percentage of cells that were vacuolated was determined.

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membrane (41, 42). Arf6 and Rac1 colocalize in these Most interestingly, Arf6 is required for trafficking of Rac1 compartments, which also contain nucleotide exchange from endosomes to the plasma membrane, so that when factors (ARNO and DOCK180/Elmo) and effectors active Rac1(Q61L) is expressed together with inhibitory (arfaptin-2/POR1 and PI4P 5-kinase) for both proteins. Arf6(T27N), both proteins accumulate in vacuole-like

FIGURE 4. Blocking Rac1 activation in glioblastoma cells prevents Ras-induced vacuolization. A, the stable cell line, U251-C18, was incubated for 3 d in the presence or absence of doxycycline (1 μg/mL), with or without the Rac1 inhibitor, EHT 1864 (25 μmol/L). Inducible expression of myc-H-Ras(G12V) was verified by Western blot analysis. B, Rac activation assays were done on cells subjected to each condition. The results are the mean (±SD) of three independent determinations done on separate cultures. The increase in active Rac1 in the cells expressing H-Ras(G12V) (+Dox alone) was significant at P < 0.001 (*) compared with the cells not expressing Ras (−Dox). The decrease in active Rac1 in the cultures expressing H-Ras(G12V) in the presence of EHT 1864 (+Dox, +EHT 1864) was significant at P < 0.001 (**) compared with the corresponding control (+Dox, −EHT 1864). The decrease in the basal level of active Rac1 in the −Dox cultures treated with EHT 1864 was significant at P < 0.05 (***). C and D, at the same time that cultures were harvested for the Rac activation assays, parallel cultures were examined by phase contrast microscopy to determine the percentage of cells that were vacuolated. The results of three separate experiments (mean ± SD) are shown. The suppression of vacuolization by the Rac inhibitor in the cultures expressing H-Ras(G12V) (+Dox, +EHT 1864) was significant at P < 0.0001 (*) compared with the cells expressing H-Ras(G12V) without the Rac inhibitor (+Dox, −EHT 1864). E, uptake of

dextran-AF568 was measured in the +Dox cells expressing myc-H-Ras(G12V), with or without EHT 1864. The results are the mean ± SD of separate determinations on three cultures. The decrease in dextran uptake in the cells treated with the Rac inhibitor was significant at P < 0.04 (*).

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structures in HeLa cells (36). A recent study has shown that Arf6 also plays a role in the trafficking of macropinosomes induced by Ras(G12V) in HeLa cells, and that perturbation of Arf6 function leads to the accumulation of CIE- derived vacuoles that contain Ras (43). For these reasons, we hypothesized that cytoplasmic vacuolization induced by H-Ras(G12V) in glioblastoma cells might entail changes in the activation state of Arf6, in addition to the activation of Rac1. To test this hypothesis, we used the U251-C18 cell line to determine if doxycycline-mediated induction of H-Ras (G12V) expression and cellular vacuolization were accompanied by changes in the amount of active Arf6. The latter was assessed in pull-down assays using a fusion protein, GST-GGA3, which interacts specifically with the GTP- bound form of Arf6. The results showed that the relative amount of active Arf6 decreased by ∼50% in the cells expressing H-Ras(G12V) (Fig. 7A and B), contrasting with the increase in Rac1 activation observed under identical conditions (Fig. 4A and B). To determine if the decrease in active Arf6 in the cells expressing H-Ras(G12V) was specifically related to the activation of Rac1, we added the Rac inhibitor, EHT 1864, at the same time that expression of H-Ras(G12V) was induced by the addition of doxycycline. By itself, EHT 1864 had no significant effect on the baseline level of active Arf6 in cells that were not expressing active H-Ras (−Dox; Fig. 7B). However, the Rac inhibitor prevented the decrease in active Arf6 (Fig. 7B) and the accumulation of vacuoles (Fig. 7C) in the cells expressing H-Ras(G12V). These results strongly suggest that the decline in the amount of active Arf6 that occurs in cells expressing H-Ras(G12V) is caused by the increase in the amount of active Rac1. To rule out the possibility that the reciprocal changes in Rac1 and Arf6 which occur in response to the expression of H-Ras(G12V) might be a unique feature of the U251 glioblastoma cell line, we compared the effects of retrovirus-mediated H-Ras(G12V) expression in U251 cells side-by-side with U20S human osteosarcoma cells (Supple- mentary Fig. S2). The results showed that the response of the osteosarcoma cell line was even more dramatic than the glioblastoma cell line. That is, Ras(G12V) expression caused a 10-fold increase in the amount of active Rac1 and a 60% decrease in the amount of active Arf6, coin- ciding with the accumulation of cytoplasmic vacuoles that incorporated the fluid-phase tracer, Lucifer yellow.

Rac1 affects Arf6 via the Arf GAP, GIT1 We next addressed the question of how the activation of Rac1 might result in a shift in the nucleotide state of Arf6 FIGURE 5. Expression of dominant-negative Rac1 blocks the induction towardtheinactiveGDP-boundform.ActiveRac of vacuolization by active H-Ras. U251 cell lines capable of conditional expression of myc-H-Ras(G12V), FLAG-Rac1(T17N), or both GTPases operate through a large number of downstream constructs, were generated using the pRetroX-Tight-Pur Tet-On system effectors to influence actin dynamics, cell migration, gene as described in Materials and Methods. A, phase contrast microscopy expression, cell survival, and endocytosis (44). However, of the cells after 2 d with or without doxycycline in the medium. only a few Rac1 effectors offer potential connections with B, immunofluorescence microscopy shows expression of myc-H-Ras (G12V) and/or FLAG-Rac1(T17N) only in the presence of doxycycline. Arf6. Arfaptin-2/POR1 is a mediator of actin dynamics The percentage of vacuolated cells in each culture was determined by and membrane ruffling that can bind to both Rac1 and scoring only the cells with visible expression of the myc and/or FLAG Arf6 (45, 46). However, arfaptin-2 binds preferentially to constructs. The results are based on counting a minimum of 70 cells.

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the inactive GDP form of Rac1 (47) and has no known To explore this possibility, we used the stable U251- effect on Arf6 GTP hydrolysis. A more likely candidate Rac1(G12V)tet cell line in which constitutively active to link Rac1 to the observed decrease in GTP-bound myc-Rac1(G12V) is conditionally expressed in the pres- Arf6 would be an Arf6 GAP that could interact with ence of doxycycline, resulting in the induction of methuo- Rac1. Among the large family of Arf GAPs (48), GIT1 sis (15). As shown in Fig. 8A, when myc-Rac1(G12V) was (also known as Cat-1 or p95-APP1) stands out because induced in these cells, immunoprecipitation of the active it can associate with both Rac1 and Rac3. Rac1-GDP is myc-Rac1(G12V) protein revealed that endogenous known to associate with GIT1 through its interaction with GIT1 was associated with it. If doxycycline was omitted, the PAK-interacting Rho exchange factor, βPIX. This in no detectable myc-Rac1(G12V) was expressed, and the turn affects the association of the GIT1/PIX complex with immunoprecipitate did not contain GIT1, ruling out non- paxillin, with consequences for cell-matrix adhesion and specific pull down of GIT1 with the myc antibody. Addi- endosomal trafficking (49-51). However, evidence from tionally, when the same experiment was done with U251 a recent study indicates that the GTP forms of Rac1 and cells expressing myc-H-Ras(G12V) instead of myc-Rac1 especially Rac3 could also interact with GIT1 (52). In the (G12V), no GIT1 was detected in the myc-H-Ras case of Rac3, interaction of the GTP-bound form with (G12V) immunoprecipitate (Fig. 8B). These observations GIT1 was direct, and results in a reduction of Arf6 activity, indicate that the active form of Rac1 is able to associate presumably through stimulation of the GAP function of with GIT1 in glioblastoma cells. GIT1 (52). We hypothesized that if Rac1-GTP interaction To assess the functional importance of GIT1 for the with GIT1 has the same effect, GIT1 might be a key induction of methuosis, we generated two stable U251 cell mediator of the Rac1-dependent decline in active Arf6. lines in which expression of GIT1 was strongly suppressed

FIGURE 6. Suppression of Tiam1 or Eps8 expression does not prevent Ras-induced vacuolization of glioblastoma cells. A, shRNA- mediated knockdown of Tiam1 or Eps8 expression was done as described in Materials and Methods. Western blots of whole cell lysates show that the specific shRNAs reduced the expression of Tiam1 or Eps8 by >90% compared with the respective controls. The loading controls, α-tubulin and LDH, confirm that comparable amounts of cell protein were applied to the lanes. B, when cells were grown in the presence of doxycycline to induce expression of myc-H-Ras(G12V) (bottom blots), there was no decrease in vacuolization of the cells lacking Tiam1 or Eps8, compared with the control. Vacuolization was assessed by phase contrast microscopy and quantified as described in Materials and Methods.

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by shRNAs targeting different sequences in the GIT1 mRNA (Fig. 9A). As a control, we used a stable U251 cell line expressing shRNA against GFP. We have previously observed that myc-Rac1(G12V) could induce a methuosis phenotype that is essentially identical to that induced by myc-H-Ras(G12V) (ref. 15 and Fig. 1). Therefore, to determine if the interaction of active Rac1 with GIT1 is required for the decline in the pool of active Arf6 and the induction of vacuolization, we introduced myc-Rac1 (G12V) into the control and GIT1 knockdown cell lines by retroviral transduction (Fig. 9). In contrast with published studies in which suppression of GIT1 caused cell rounding and detachment of neuronal cells (31), the glioblastoma cell lines lacking GIT1 exhibited cell morpho- logies and growth rates that were comparable to the control cells. When myc-Rac1(G12V) was expressed in these cell lines, the control cell line showed a ∼50% reduction in the amount of active Arf6, compared with the same cells infected with empty retroviral vector (Fig. 9B). In contrast, expression of Rac1(G12V) in the two GIT1 KD cell lines caused a negligible reduction in the amount of active Arf6 (Fig. 9B). The preservation of active Arf6 in the GIT1 KD cells was associated with a reduced amount of cytoplasmic vacuolization and cell death in response to expression of Rac1(G21V) (Fig. 9C-E). These findings support the idea that the reduction in the amount of Arf6-GTP caused by activation of Rac1 could be mediated, at least in part, through stimulation of the GAP function of GIT1. They also reinforce the notion that the reduction in the pool of active Arf6 is an important contributing factor to the endosomal trafficking defects that underlie the methuosis phenotype.

Discussion

The prognosis for patients with glioblastoma remains poor, despite efforts to improve surgical, radiological, and chemotherapeutic treatment strategies. A major problem is that glioblastoma cells harbor genetic mutations that render them resistant to apoptosis (53, 54). The discovery of cell death mechanisms that do not depend on activation of the classical mitochondrial and death receptor–mediated FIGURE 7. Expression of activated H-Ras causes a Rac1-dependent decrease in the amount of active Arf6, which is essential for vacuolization apoptotic pathways could present new opportunities of glioblastoma cells. A, U251-C18 cells were incubated for 3 d in the to slow the progression of these aggressive tumors. In this presence or absence of doxycycline (1 μg/mL), with or without the Rac1 report, we have begun to define the signaling pathways inhibitor, EHT 1864 (25 μmol/L). Inducible expression of myc-H-Ras underlying a nonconventional form of cell death termed (G12V) was verified by Western blot analysis. B, Arf6 activation assays were done on cells subjected to each condition, with active Arf6 methuosis, which can be triggered by ectopic expression normalized to total Arf6 as described in Materials and Methods. The of constitutively activated Ras in a broad spectrum of glio- results are the mean (± SD) of three independent determinations done on blastoma cell (15), as well as gastric carcinoma cells (14) separate cultures. The decrease in active Arf6 in the cells expressing and osteosarcoma cells lines (Supplementary Fig. S2). H-Ras(G12V) (+Dox alone) was significant at P < 0.01 (*) compared with − The hallmark cytopathologic feature of methuosis is the the cells not expressing Ras ( Dox) or the cells incubated with the Rac inhibitor, EHT 1864. Representative immunoblots of the raw Arf6 GTP displacement of much of the cellular cytoplasmic space by pull-downs are shown below the bar graph. C, at the time that cultures fluid-filled vacuoles derived from macropinosomes or CIE were harvested for the Arf6 activation assays, parallel cultures were (15). Macropinosomes are formed when membrane ruffles examined by phase contrast microscopy to determine the percentage of (lamellipodia) enclose pockets of extracellular fluid and cells that were vacuolated. The results of three separate experiments (mean ± SD) are shown. The suppression of vacuolization caused by are internalized (55, 56). We previously showed that cells addition of the Rac inhibitor to cultures expressing H-Ras(G12V) undergoing methuosis in response to the expression of (+Dox, +EHT 1864) was significant at P < 0.0001 (*) compared with the cells constitutively active H-Ras(G12V) exhibit increased expressing H-Ras(G12V) without the Rac inhibitor (+Dox, −EHT 1864).

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Our previous studies have indicated that methuosis involves dysfunction in other steps of the endosomal trafficking pathway beyond the initial stimulation of macropinocytosis. During methuosis, fluid-filled endocytic vesicles tend to accumulate instead of being recycled or merged with lysosomes (15). Our current results show that a decline in the pool of active Arf6 is important for vacuolization induced by activation of Rac1 (Fig. 7; Supplementary Fig. S2). It is likely that this is due to loss of function of Arf6-GTP, rather than gain of function of Arf6-GDP because overexpression of the GDP-locked Arf6(T27N) does not induce vacuolization in U251 cells.1 We also show that the decline in active Arf6 requires the presence of GIT1 (Fig. 9), an Arf6 GAP that can interact with Rac1 (Fig. 8). Because activation of Arf6 is important for recycling macropinosomes back to the cell surface (58), these findings suggest a model wherein chronic activation of Rac1 plays a dual role, stimulating the formation of in- coming macropinosomes while simultaneously impeding FIGURE 8. Active Rac1 interacts with the Arf6 GAP, GIT1. A, 3 d after macropinosome recycling by decreasing the pool of adding doxycycline to induce expression of myc-Rac1(G12V) in a Arf6-GTP. The resulting overload of fluid-filled vesicles stable U251 cell line, cells were lysed and the myc-tagged protein was collected on agarose beads conjugated with anti-myc antibody. Samples may promote their abnormal fusion to generate progres- of whole-cell lysate and proteins eluted from the myc-agarose beads sively larger vacuoles which, in the absence of effective were subjected to Western blot analysis with monoclonal antibodies lysosomal clearance, fill the cytoplasm and disrupt the against GIT1 or myc. Immunoprecipitation of lysate from cells that did not cells (Fig. 10). The postulated block in trafficking receive doxycycline was done to control for nonspecific precipitation of GIT1. B, the same experiment was done with stable U251 cells between clathrin-independent late endosomes and induced to express myc-H-Ras(G12V) to establish that GIT1 does not lysosomes is supported by the observation that vacuoles associate with activated H-Ras. labeled with fluorescent dextran in glioma cells expressing H-Ras(G12V) do not merge with LysoTracker-positive macropinocytotic uptake of fluid phase tracers (15). How- compartments (15). Furthermore, although the Ras- ever, unlike normal macropinosomes, which either recycle induced vacuoles are not sufficiently acidic to sequester back to the plasma membrane or merge with lysosomal LysoTracker or acridine orange, and they do not contain compartments (57, 58), labeled vesicles internalized into lysosomal enzymes like cathepsin B, some of them do cells undergoing methuosis do not seem to fuse with lyso- acquire LAMP1 (15) and Rab 7 (Supplementary somes or release their contents at the cell surface (15). Fig. S3). These features are consistent with arrest at a late Thus, large vacuoles accumulate and eventually cause a endosomal stage. The molecular basis for the latter traf- necrosis-like rupture of the cell that cannot be prevented ficking defect remains to be defined, but Rac1 modula- with caspase inhibitors. tion of Rab7, which functions in trafficking between late The present studies establish that the methuosis phe- endosomes and lysosomes, seems to be a likely possibility. notype induced by overexpression of activated H-Ras In support of this concept, Sun et al. (64) have reported a depends on downstream activation of the Rac1 GTPase direct interaction between Rac1 and Rab7 in osteoclasts. (Figs. 2–5). Rac1 is widely recognized as an important reg- Very recently, Frasa et al. (65) described a novel protein ulator of actin dynamics, integrin-mediated cell adhesion, named Armus, which associates with the GTP-bound and cell migration (44, 59), but there is also abundant form of Rac1 and inactivates Rab7 through its COOH- evidence implicating Rac1 in the control of endocytic ves- terminal GAP domain. Based on this observation, we pre- icle trafficking (60, 61). Active Rac1 seems to affect endo- dict that chronic activation of Rac1 might interfere with cytosis at multiple levels. On one hand, Rac1 interacts Rab7-mediated trafficking events at the interface between with synaptojanin-2 and downregulates clathrin-mediated late endosomes and lysosomes (Fig. 10). Future investiga- receptor endocytosis (62, 63). However, the active form tion of the role of signaling between Rac1 and Rab7 pro- of Rac1 has the opposite effect on clathrin-independent mises to provide additional insights into the molecular endocytosis, stimulating membrane ruffling and promot- basis for endosomal vacuolization in methuosis. ing macropinocytosis (22). Our results strongly suggest At first glance, our finding that inactivation of Arf6 that in glioblastoma cells in which overexpression of acti- contributes to vacuolization of cells expressing active Ras vated H-Ras drives the continuous activation of endoge- might seem contradictory to the observations of Porat- nous Rac1, the chronic stimulation of macropinocytosis Shliom et al. (43), who found that active Arf6(Q67L) by Rac1 is an important factor in the biogenesis of the large cytoplasmic vacuoles that eventually compromise the integrity of the cell. 1 Unpublished observation.

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induces the formation of vacuoles in cells expressing H-Ras and PI(3,4,5)P3, but have not yet acquired Rab5 or Rab7, (G12V). However, the vacuoles induced by active Arf6 orig- which mark early and late endosomes, respectively (43). In inate at a different stage of the endocytic pathway than those contrast, vacuoles that form when H-Ras(G12V) activates induced by inactivation of Arf6. Specifically, in cells coexpres- endogenous Rac1, with concordant inactivation of Arf6, have sing H-Ras(G12V) with Arf6(Q67L), vacuoles arise because characteristics of a late endosomal compartment that has ac- nascent macropinosomes are trapped at an early stage after quired LAMP1 (15) and Rab7 (Supplementary Fig. S3). This internalization, resulting in vesicles that contain PI(4,5)P2 would be consistent with our model (Fig. 10) wherein CIEs

FIGURE 9. Suppression of GIT1 expression prevents the Rac1(G12V)-mediated decrease in active Arf6 and protects glioblastoma cells from methuosis. Stable cell lines were generated from U251 cells infected with lentivirus encoding shRNA against GFP (control) and two different shRNAs against GIT1 (GIT1 a and b). A, the control and GIT1 knockdown cell lines were infected with retrovirus encoding myc-Rac1(G12V), and 3 d after infection, the cells were subjected to Western blot analysis to check expression of myc-Rac1(G12V) and GIT1. B, on the 4th d after infection, the control and GIT1 knockdown cell lines were assayed for the amount of active Arf6 as described in Materials and Methods. The results show the amount of active Arf6 in the cells expressing myc-Rac1(G12V), expressed as a percentage of the value obtained for the same cell line infected with empty retroviral vector (mean ± SD for three experiments). The differences between each of the GIT1 knockdown cell lines (*) and the control cell line were significant at P < 0.004. C and D, on the 4th d after infection with empty or myc-Rac1(G12V) retroviral vectors, the control and GIT1 knockdown cell lines were examined by phase contrast microscopy to determine the percentage of cells that were vacuolated. The results of three separate experiments (mean ± SD) are shown in the bar graph. The suppression of Rac1-induced vacuolization in the GIT1 knockdown cell lines (*) was significant at P < 0.0001 compared with the control cell line. E, on the 5th d after infection with empty or myc-Rac1(G12V) lentiviral vectors, the control and GIT1 knockdown cells were assayed for cell viability using MTT. Assays were done on quadruplicate cultures in a 96-well plate, and the results for the cells infected with myc-Rac1(G12V) retrovirus were expressed as a percentage of the same cells infected with empty vector. The increased viability observed in each of the GIT1 knockdown cell lines was significant at P < 0.007 relative to the shRNA control cell line.

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FIGURE 10. A hypothetical model for Ras-induced methuosis suggested by the present findings. (1) Expression of constitutively activated H-Ras reaches a threshold sufficient to stimulate the activity of an unidentified Rac1 GEF. (2) The resulting increase in the pool of active Rac1 enhances macropinocytosis. (3) As the pool of active Rac1 increases beyond normal physiologic levels, Rac1-GTP associates with GIT1 and stimulates Arf6 GAP activity. The consequent decrease in the pool of active Arf6 impairs recycling of CIEs. (4) CIEs derived from macropinosomes acquire some characteristics of late endosomes (Rab7 and LAMP1), but fail to merge with lysosomes, due to trafficking defects at the late endosome/lysosome boundary. One possible mechanism for a block in lysosomal fusion could be Rac1 stimulation of the Rab7 GAP, Armus, resulting in inactivation of Rab7, as recently described by Frasa et al. (65). (5) The accumulated CIE and late endosomal vesicles coalesce to form progressively larger vacuolar structures, which ultimately displace most of the cytoplasm and lead to cell detachment from the substratum and disruption of cell membrane integrity.

fail to recycle and arrest at a stage before lysosomal fusion, but docytic trafficking and cell death through alterations of Rac1 after the Rab5/Rab7 conversion (66). and Arf6 in others. The answer might lie, at least in part, in The ability of activated Ras to stimulate macropinocytosis cell type–specific variations in the expression of nucleotide has been recognized for a number of years (67, 68), but the exchange factors that can link the activation of Ras to Rac1. connection between dysfunctional Ras-induced macropino- In this regard, our finding that neither Tiam1 nor Eps8 was cytosis and cell death in specific types of cancer cells has only essential for Ras to induce methuosis raises the possibility recently been appreciated. The present study implicates the that other poorly characterized nucleotide exchange factors Rac1 and Arf6 GTPases as important downstream compo- (e.g., Tiam2/Stef) or novel signaling pathways might play nents in this process. A major unanswered question is why such a role in glioblastoma cells. the expression of constitutively activated Ras mutants can Another unsolved puzzle is why Rac1 activation can pro- promote cell proliferation and survival through classical mi- mote cell motility and invasiveness (59, 69, 70), yet can togen-activated protein kinase and PI3K signaling pathways also trigger cytopathologic changes in vesicular trafficking in some types of cells, but can also cause disruptions of en- that lead to cell death. Our results suggest that a key to

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understanding the paradoxical effects of Rac1 might lie in attention. However, the recent finding that high-grade the balance between active Arf6 and Rac1. Under normal human gliomas and glioma cell lines have elevated levels physiologic conditions, Arf6 cycles between the inactive of Arf6 compared with low-grade tumors or normal brain GDP state and the active GTP state. Arf6-GTP promotes tissue (74) suggests that significant variations in Arf6 signal- cell adhesion and migration by facilitating translocation of ing mechanisms may occur in some types of cancer cells. Rac1 to the leading edge of the cell membrane and stim- Our initial characterization of methuosis has focused ulating activation of Rac1 via nucleotide exchange factors on cells in which this form of death can be triggered by like the DOCK180/Elmo complex (71) or Kalirin (72). ectopic expression of activated Ras or Rac1. However, we The multifunctional scaffold protein, GIT1, also seems believe that methuosis may represent just one example of a to play an important role in Rac1 trafficking to points of larger group of cytopathologies characterized by dysfunc- cell adhesion (49, 73). GIT1 can associate with both the tional trafficking and vacuolization of early or late endo- GDP and GTP forms of Rac1, but the indirect interaction somal compartments. For instance, in cells exposed to with Rac1-GDP through βPIX seems to predominate (52). the H. pylori VacA toxin (75) or the PI3K inhibitor, wort- However, based on the coimmunoprecipitation of Rac1 mannin (76), cells are disrupted by the accumulation of (G12V) with GIT1 (Fig. 8), we speculate that in cells large vacuoles that arise from the reorganization of late where overexpression of H-Ras(G12V) drives constitutive endosomes and lysosomes. Interestingly, the induction of activation of endogenous Rac1, or in cells where active vacuoles by VacA seems to depend on Rac1 signaling Rac1(G12V) is highly expressed, a direct interaction be- because it is potentiated by expression of activated Rac1 tween the GTP form of Rac1 and GIT1 might be favored and inhibited by dominant-negative Rac1 (18). In another over the “normal” interaction with Rac1-GDP. Direct in- case, cultured cells overexpressing a peripheral nerve teraction between Rac3-GTP and GIT1 has been shown to myelin protein, Gas3/PMP22 (growth arrest–specific 3/ reduce Arf6 activity (52). Our observation that the Rac1 peripheral myelin protein 22), have been found to accu- (G12V)-induced decrease in active Arf6 was prevented mulate vacuoles derived from an Arf6-dependent noncla- by knockdown of GIT1 (Fig. 9) suggests that Rac1-GTP thrin endosome recycling compartment (77). These may have a similar ability to reduce the pool of active Arf6 observations suggest that perturbation of Rac1- and by stimulating the GAP activity of GIT1. Arf6-dependent trafficking pathways might be a common It is important to note that although our model (Fig. 10) mechanism in various cytopathologies associated with might apply to glioblastoma cells and other types of cancer cytoplasmic vacuolization and necrosis-like cell death. cells (osteosarcoma and gastric carcinoma), it remains un- Ultimately, as more is learned about the relevant signaling clear if all cell types have the potential to respond to expres- pathways, the identification of drug-like compounds that sion of activated Ras or Rac1 by undergoing vacuolization can trigger methuosis in tumor cells may provide new and cell death. We previously reported that in some types opportunities for treatment of cancers that do not respond of cells (HEK293 and human skin fibroblasts), expression well to conventional agents that work by inducing apoptosis. of H-Ras(G12V) did not elicit the methuosis phenotype (15). However, the results obtained in the present study Disclosure of Potential Conflicts of Interest with graded H-Ras(G12V) expression (Fig. 3) raised the possibility that the lack of response previously seen in some No potential conflicts of interest were disclosed. cell lines could have been due to the level of Ras expression remaining below the threshold needed for Rac1 activation. Acknowledgments We have since revisited this issue using a retroviral vector to obtain nearly identical levels of H-Ras(G12V) overexpres- We thank Dr. Laurent Désiré and ExonHit Therapeutics for providing EHT sion in U251, U20S, and HEK293T cells, and found that 1864, and Dr. Amy Wilson-Delfosse for the Rac1(G12V) construct. under these conditions, the HEK 293 cells could in fact be induced to undergo morphologic changes typical of Grant Support methuosis (Supplementary Fig. S4). Other factors that NIH grant R01 CA115495 (W.A. Maltese). might influence the responses of particular cell types might The costs of publication of this article were defrayed in part by the payment of include the expression level of GIT1 relative to other Arf6 page charges. This article must therefore be hereby marked advertisement in GAPs or the expression level of Arf6 itself. The question of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tissue-specific variations in these proteins in different types Received 03/03/2010; revised 07/27/2010; accepted 08/05/2010; published of normal and transformed cells has not received much OnlineFirst 08/16/2010.

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Induction of Nonapoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

Haymanti Bhanot, Ashley M. Young, Jean H. Overmeyer, et al.

Mol Cancer Res Published OnlineFirst August 16, 2010.

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