FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2

FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2 Koji Tsutsumi, Ayumi Nohara, Taiki Tanaka, Moe Murano, Yurina Miyagaki, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3219907/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Dec, 2023 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that forms the two different protein complexes, known as mTORC1 and mTORC2. mTOR signaling is activated in a variety of tumors, including glioma that is one of the malignant brain tumors. FilGAP (ARHGAP24) is a negative regulator of Rac, a member of Rho family small GTPases. In this study, we found that FilGAP interacts with mTORC1/2 and is involved in tumor formation in glioma. FilGAP interacted with mTORC1 via Raptor and with mTORC2 via Rictor and Sin1. Depletion of FilGAP in KINGS-1 glioma cells decreased phosphorylation of S6K and AKT. Furthermore, overexpression of FilGAP increased phosphorylation of S6K and AKT, suggesting that FilGAP activates mTORC1/2. U-87MG, glioblastoma cells, showed higher mTOR activity than KINGS-1, and phosphorylation of S6K and AKT was not affected by suppression of FilGAP expression. However, in the presence of PI3K inhibitors, phosphorylation of S6K and AKT was also decreased in U-87MG by depletion of FilGAP, suggesting that FilGAP activates mTORC2 in PI3K-independent manner. Finally, we showed that depletion of FilGAP in KINGS-1 and U-87MG cells significantly reduced spheroid growth. These results suggest that FilGAP may contribute to tumor growth in glioma by regulating mTORC1/2 activities. Biological sciences/Cell biology/Cell growth/Tor signalling Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that is highly conserved from yeast to mammals belonging to the PI3K-related protein kinases family. mTOR forms functionally distinct protein complexes, mTORC1 and mTORC2. mTORC1 has Raptor, and mTORC2 has Rictor and Sin1 as specific subunits respectively 1 – 4 . mTORC1 is activated by amino acids, and regulates protein synthesis and autophagy by phosphorylating downstream substrates, such as S6 kinase and 4EBP1 5–8 . mTORC2 is primarily activated by growth factors such as insulin, and regulates cell proliferation, cell survival, and cell migration by phosphorylating AKT, PKC and other substrates 9 – 14 . While the detailed mechanism of mTORC1 activation has been clarified, the molecular mechanism of mTORC2 activation remains unclear. Since new regulators of mTORC1 and mTORC2 activity have been reported even recently, the regulatory pathway of mTOR is considered to be very complex 14 – 20 . mTOR signaling is activated in a variety of tumors 4 , 21 , 22 . In particular, in glioma, one of the malignant brain tumors, activation of the mTOR pathway promotes cell proliferation and invasion, and contributes to patient poor prognosis 23 – 26 . Rho small GTPases cycle between active GTP-bound and inactive GDP-bound state, and act as key regulators of actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) activate Rho GTPase by catalyzing the exchange of GDP for GTP. While GTPase-activating proteins (GAPs) stimulate the intrinsic GTPase activity and inactivate them 27 . FilGAP (also known as ARHGAP24) is a Filamin A-binding protein, and Rac-specific GAP 28 – 31 . FilGAP suppresses leading edge protrusion and promotes cell retraction by inhibiting Rac and also regulates epithelial cell-cell adhesion and tissue morphogenesis 32 – 35 . In addition, it has been suggested that the expression level of FilGAP is associated with malignant transformation of cancer and is also involved in invasive metastasis and tumorigenesis 36 – 41 . However, the contribution of FilGAP to cancer development is thought to be different among cancer types, and more detailed mechanisms need to be elucidated. In this study, we found that FilGAP interacts with mTORC1 and mTORC2. We showed that Raptor, Rictor and mTOR were coprecipitated with FilGAP. Depletion of FilGAP expression in KINGS-1 gliomas had an inhibitory effect on S6K and AKT phosphorylation, while overexpression of FilGAP had a promotive effect, suggesting that FilGAP activates mTORC1 and mTORC2. A short FilGAP variant lacking partial PH domain was highly expressed in gliomas, and this short variant also contributed to mTORC1 and mTORC2 activation. Depletion of FilGAP did not affect mTOR activity in U-87MG, a highly malignant glioblastoma, but significantly decreased AKT phosphorylation in the presence of a PI3K inhibitor. Finally, we showed that depletion of FilGAP inhibited spheroid formations in KINGS-1 and U-87MG as well as mTOR inhibition. These results suggest that FilGAP interacts with mTORC1 and mTORC2 and may positively regulate their activity to promote tumorigenesis in gliomas. Results FilGAP interacts with mTORC1 and mTORC2 Recently, it has been reported that mTORC2 interacts with an actin filament crosslinking protein, Filamin A in glioblastoma cells 18 , 26 . Furthermore, mTOR has been reported to interact with various Rho family GTPases and their regulators 14 , 15 , 42 , 43 . Thus, we considered the possibility that FilGAP interacts with mTORC2 through Filamin A. To examine whether FilGAP interacts with mTOR complex, we transfected Raptor or Rictor with or without FilGAP in HEK293T cells and performed coimmunoprecipitation assay (Fig. 1A, B). Both Raptor and Rictor were coprecipitated with FilGAP, suggesting that FilGAP interacts with Raptor and Rictor. Rictor was also coprecipitated with FilGAP V734Y, Filamin A-binding deficient mutant, suggesting that this interaction is independent of Filamin A. Further, we investigated whether mTOR components in HEK293T cell lysates were pulled down with GST-FilGAP (373-748aa) (Fig. 1C). mTOR, Raptor and Rictor were co-precipitated with GST-FilGAP. This result suggests, that FilGAP may interact with both mTORC1 and mTORC2. The mTOR complexes are disrupted in the buffer with non-ionic detergent such as TritonX-100, but can be maintained in the buffer containing amphoteric detergent CHAPS 3 . Therefore, we prepared cell lysates with TritonX-100 or CHAPS and performed GST-pulldown assay (Fig. 1D). In the presence of TritonX-100, the coprecipitation with GST-FilGAP was decreased for Raptor and mTOR, but slightly increased for Rictor. We further examined the interaction of mTOR complex with FilGAP in Raptor- or Rictor-depleted cells (Fig. 1E). Knockdown of Raptor, but not Rictor, reduced the binding of mTOR to FilGAP, suggesting that most of the mTOR coprecipitated with FilGAP is mediated by Raptor. We examined whether binding to FilGAP is altered when the kinase activity of the mTOR is suppressed. Treatment of the cells with Rapamycin, mTORC1 specific inhibitor, and Torin1, mTOR inhibitor significantly decreased the amount of Raptor and mTOR coprecipitated with FilGAP. On the other hand, Rictor coprecipitated with FilGAP was rather increased by the inhibitor treatments. Similar data were observed when mTORC1 was activated by amino acids and insulin stimulation. (Fig. S1 A, B). These data suggest that the interaction of mTORC1 and FilGAP is dependent on mTORC1 activity. Identification of domains mediating FilGAP-mTORC1/2 interaction To clarify the manner of the interaction between FilGAP and mTOR complexes, we examined detailed interaction between FilGAP and mTOR components. We generated a series of FilGAP-deletion mutants and examined their binding regions to Raptor or Rictor (Fig. 2A-D, Fig. S2A, B). C-terminus deletion resulted in a substantial decrease in interaction with Raptor, suggesting that the C-terminal region of FilGAP is important for binding to Raptor (Fig. 2A, B, Fig. S2A, B). On the other hand, it is suggested that FilGAP 500-520aa and 560-570aa are important for binding to Rictor (Fig. 2C, D). We generated deletion mutants of Raptor and examined their binding to GST-FilGAP and found that it binds to NT and CT but not to MD (Fig. 2E, F, Fig. S2C). Sin1, a specific component of mTORC2, also coprecipitated with FilGAP and the N-terminus of Sin1 was important for this binding. (Fig. 2G, H, Fig. S2D). Sin1 and FilGAP also co-precipitated in binding experiments with purified proteins, suggesting that this binding is direct (Fig. 2H). FilGAP activates mTORC1/2 in glioma cells To study the role of interaction between mTORC1/2 and FilGAP, we used KINGS-1 human astrocytoma cell line, which expresses high level of FilGAP 36 . Small interference RNAs (siRNAs) targeting FilGAP were transfected to KINGS-1 cells. Depletion of FilGAP in KINGS-1 cells significantly reduced phosphorylation of S6K and AKT, which are substrates of mTORC1 and mTORC2, respectively (Fig. 3A, B). Furthermore, transfection of HA-tagged FilGAP in KINGS-1 cells increased phosphorylation of S6K and AKT, and rescued the decrease in their phosphorylation caused by depletion of FilGAP (Fig. 3C, D). These results suggest that FilGAP may activate mTORC1/2 in KINGS-1 cells. PH domain of FilGAP is important for regulation of mTORC2 activity FilGAP contains pleckstrin homology (PH), Rho GAP, and coiled-coil (CC) domains 28 . FilGAP R175A has mutation at GAP domain and lacks GAP activity. PH domain of FilGAP is required for Phosphatidylinositol 3-phosphate (PIP3)-dependent membrane localization 44 . To study whether GAP activity and PH domain of FilGAP is required for the regulation of mTORC1/2 activities, HA-FilGAP constructs (WT, R175A or DPH; Fig. 4A) resistant to FilGAP siRNA were transfected to KINGS-1 cells after depletion of endogenous FilGAP with siRNA (Fig. 4B). Although FilGAP wild-type (WT) and R175A significantly increased AKT phosphorylation, FilGAP DPH did not increase AKT phosphorylation as much as WT and R175A (Fig. 4C). On the other hand, all FilGAP constructs increased S6K phosphorylation to the same extent (Fig. 4D). These results suggest that PH domain of FilGAP is important for the regulation of mTORC2 activity. FilGAP partial PH-deleted transcript variant activates mTORC1/2 There are multiple FilGAP variants, some of which are partially or completely lacking PH domain (Fig. 5A) 45 , 46 . The expression of FilGAP variants was examined by RT-PCR analysis and variants 3 and 4 were highly expressed in gliomas (U-87MG and KINGS-1) compared to melanoma (A7) and breast cancer cells (MDA-MB-231) (Fig. 5B). To study whether FilGAP variants 3 and 4 also regulate mTOR activity, siRNA targeting specific FilGAP variant 1 was transfected to KINGS-1 cells. Knockdown of FilGAP variant 1–4 (all isoforms) and only FilGAP variant 1 were confirmed by immunoblotting (Fig. 5B). Depletion of all FilGAP variants by siRNA KD#1 decreased phosphorylation of S6K and AKT, while depletion of only FilGAP variant 1 significantly increased their phosphorylation. (Fig. 5B, C). In addition, depletion of only FilGAP variant 1 tended to increase the expression levels of variants 3 and 4. To further investigate the effect of FilGAP variant 3 on the activities of mTORC1/2, HA-FilGAP constructs (96-748aa; variant 3) resistant to FilGAP siRNA was transfected to KINGS-1 cells after depletion of FilGAP with siRNA. FilGAP (96-748aa) increased mTORC1/2 activities to the same extent as WT (Fig. 5D, E). Interestingly, FilGAP 96–748 aa showed increased coimmunoprecipitation with Rictor rather than WT (Fig. 5F). These results suggest that FilGAP variant 3, 4 may have a higher or equal ability to activate mTORC1/2 compared to variant 1. Give that ΔPH (131-748aa) cannot increased mTORC2 activity, 96–130 aa of FilGAP may be important to activate mTORC2. FilGAP is involved in PI3K-independent mTORC2 activation in glioblastoma cells Gliomas are classified from grade II to grade IV according to histological and genetic diagnosis 47 . Grade IV glioblastoma is the most aggressive and has a very poor prognosis with a 2-year survival rate of less than 30%. To investigate the relationship between the mTORC1/2 regulation by FilGAP and grade of glioma malignancy, we compared mTORC1/2 activities and the expression levels of FilGAP in KINGS-1 and U-87MG, grade III glioma and grade IV glioblastoma, respectively. Phosphorylation of S6K and AKT were higher in U-87MG than in KINGS-1, suggesting that mTORC1 and mTORC2 were more active in U-87MG. (Fig. 6A). The expression levels of FilGAP was higher in KINGS-1 cells than in U-87MG (Fig. 6B). To study whether FilGAP regulates mTORC1/2 activities in U-87MG cells as observed in KINGS-1, siRNAs targeting FilGAP was transfected to U-87MG and examined phosphorylation of AKT and S6K. Depletion of FilGAP in U-87MG cells did not affect phosphorylation of S6K and AKT, unlike in KINGS-1 cells (Fig. 6C, D). Since mTORC1/2 activities are very high in U87MG, we postulated that the contribution of FilGAP to mTORC1/2 activities may be small. PI3K signaling, one of the upstream activator of mTOR, is known to be activated in glioblastoma by genetic mutations or deletions. So, we examined the effect of FilGAP depletion in U-87MG cells in the presence of PI3K inhibitor (LY294002). PI3K inhibition greatly reduced phosphorylation of AKT and almost completely abolished phosphorylation of S6K. And depletion of FilGAP significantly decreased AKT phosphorylation in U-87MG cells in the presence of PI3K inhibitor (Fig. 6E, F). These results suggest that FilGAP may be involved in PI3K-independent mTORC2 activation in glioblastoma cells. FilGAP regulate spheroid growth in KINGS-1 and U-87MG cells mTOR regulates glioma survival, proliferation, and invasion. To study the role of mTORC1/2 regulation by FilGAP in glioma, we examined whether inhibition of mTOR or depletion of FilGAP affect tumorigenesis using 3D spheroid model of KINGS-1 and U-87MG cells. When KINGS-1 and U-87MG were seeded on low-attachment U-bottom plates, the spheroid size increased with the number of days in culture. We used PI3K inhibitor (LY294002), mTOR inhibitor (Torin1), and mTORC1 inhibitor (Rapamycin) to inhibit mTOR activity. All inhibitors treatment reduced spheroid diameter in both KINGS-1 and U-87MG cells, indicating that mTOR activity is critical for spheroid growth (Fig. 7A, B). Depletion of FilGAP also reduced spheroid diameter in both KINGS-1 and U-87MG (Fig. 7C, D), suggesting that FilGAP promotes spheroid growth in glioma as well as mTOR. Finally, we examined the effects of a combination of mTOR inhibition and suppression of FilGAP expression on spheroid growth. The combination of mTOR inhibitor treatment and suppression of FilGAP expression significantly reduced spheroid size compared to either treatment alone (Fig. 7E, Fig. S3). These data suggest that the combination of mTOR inhibitor treatment and suppression of FilGAP expression has a potent inhibitory effect on tumorigenesis. Discussion In this study, we found that FilGAP interacts with mTORC1/2. FilGAP seems to interact with mTORC1 through Raptor and with mTORC2 through Rictor or Sin1 (Fig.1 and 2). We also found that FilGAP regulates mTORC1/2 activities in glioma cells. PH domain (1-130aa) of FilGAP, especially the region 96-130aa, is important for mTORC2 activation (Fig. 6). In addition, we showed that FilGAP may be involved in PI3K-independent mTORC2 activation in glioblastoma. Finally, we showed that the suppression of FilGAP expression and mTOR inhibition attenuated spheroids growth in both KINGS-1 and U-87MG. Taken together, these results suggest that FilGAP may regulate tumor growth in gliomas through the regulation of mTORC1/2 activities. We showed that FilGAP interacts with mTORC1/2 and regulates their activities. However, it is still unclear how FilGAP regulates mTORC1/2 activities. mTORC1 is activated by amino acids and other nutrients though the Rheb and Rag GTPases 4 . On the other hand, mTORC2 is activated primarily by growth factors through PI3K signaling 48 . One possibility is that FilGAP may promote complex formation of mTORC1 and mTORC2. In addition, there is a crosstalk pathway between mTORC1 and mTORC2 that regulates each other's activity 49 . Since FilGAP binds to both mTORC1 and mTORC2, it may be involved in the regulation of mTORC1 and mTORC2 cross-talk. Future clarification of the detailed pathway of mTOR activation by FilGAP may shed light on the very complex regulatory mechanism of mTOR pathway. We showed that FilGAP transcript variants partially lacking PH domain, which are highly expressed in gliomas, also activate mTORC1 and mTORC2 (Fig. 5). Further 96-130aa of FilGAP may be important for mTORC2 activation (Fig. 4, 5). FilGAP 96-748aa corresponding to variant 3 was suggested to bind Rictor more strongly than the full length (Fig. 5G). It is possible that FilGAP 1-95aa acts to interfere with the interaction with Rictor. FilGAP with PH domain can also activate mTORC2, it is possible that the binding of PIP3 to the PH domain of FilGAP affects its interaction with mTORC2. Since FilGAP 1-95aa contains Arginine 39, which is important for binding to PIP3, it is considered that FilGAP variant 3 cannot bind to PIP3 44 . Therefore, it is likely that FilGAP lacking PH domain can interact with mTORC2 in a PIP3-independent manner. Depletion of FilGAP in U-87MG, grade IV glioblastoma cell line, did not affect mTORC1/2 activities (Fig. 6). Since highly malignant glioma such as glioblastoma acquires high mTORC1/2 activities by the gene mutations, the contribution of FilGAP to mTORC1/2 activities seems to be reduced. Actually, U-87MG cells have mutations in PTEN, a negative regulator of mTOR pathway 50,51 . However, depletion of FilGAP in U-87MG decreased mTORC2 activity in the presence of PI3K inhibitors (Fig. 6E, F). This suggests that FilGAP may regulate mTORC2 activity PI3K-independent manner. PI3K-independent activation of mTOR has been reported in drug-resistant cancer cells 52 . FilGAP may be involved in the acquisition of drug resistance by cancer cells with high PI3K activity. Remarkably, glioma cells express high levels of FilGAP variant 3 and 4, which structurally appears to function in a PIP3-independent manner. FilGAP transcript variant lacking a part of PH domain may be important for PI3K-independent survival of cancer cells. This may be the reason that depletion of FilGAP suppresses spheroid growth not in KINGS-1 cells but also U-87MG cells (Fig. 7C, D). Since the low adhesion and hypoxic conditions during spheroid formation affect the PI3K pathway 53 , the regulation of mTORC1/2 activities by FilGAP might be prominently observed. This study provides FilGAP as a new PI3K-independent mTORC1/2 activator. FilGAP may become a potential therapeutic target for drug-resistant glioblastoma. It will be important issue to be solved to determine how FilGAP activates mTORC1/2, and the role of this regulation in vivo. Methods Cell culture, reagents and antibodies KINGS-1 cells (GIII astrocytoma, HSRRB) were maintained in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin and 50 mg/ml streptomycin. U-87MG (Glioblastoma, ATCC) cells were maintained in Eagle's minimal essential medium (E-MEM) supplemented with 10% FBS, 50 units/ml penicillin and 50 mg/ml streptomycin. HEK293T cells, MDA-MB-231cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. A7 cells were provided from Dr. Thomas P. Stossel (Harvard University). A7 cells were maintained in Minimum Essential Medium Eagle supplemented with 8% newborn calf serum, 2% fetal calf serum, 50 units/ml penicillin and 50 mg /ml streptomycin. Reagents, antibodies, siRNA used in this study were listed in supplementally table 1. Transfection KINGS-1 cells were transfected with plasmid DNA for 24 hours using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. HEK293T cells was transfected with plasmid DNA for 24 hours using Poly-Ethylene-Imine (PEI, polyscience). KINGS-1, U-87MG cells were transfected with FilGAP siRNA for 48 or 72 hours using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. The cells were maintained at 37°C with 5% CO₂ during the treatments. Immunoblotting Total cellular proteins were harvested using RIPA buffer [20 mM Tris‐HCl (pH7.5), 120 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mM MgCl 2 , 1 mM EDTA, 10 mM NaF, 10 mM b-glycerophosphate, Protease inhibitor Cocktail and 1 mM DTT]. Cell lysates were separated by SDS‐PAGE and transferred to the membrane for fluorescence (Millipore). The membrane was blocked with Intercept (PBS) Blocking Buffer (LI-COR) and incubated with primary antibodies. The primary antibodies were detected with secondary antibodies and FUSION SOLO S (Vilber Loumat). GST pulldown assay HEK293T cells were washed with phosphate-buffered saline (PBS), suspended with CHAPS buffer (50mM Tris-HCl [pH 7.5], 0.1M NaCl, 2mM MgCl 2 , 0.1mM EDTA [pH 8.0], 0.3% CHAPS, Protease inhibitor Cocktail (SIGMA), and 1mM DTT). The cells were disrupted and the cell lysates were prepared by centrifugation for at 15,000 rpm for 5 min at 4°C. The supernatant fluid was incubated with GST-FilGAP protein coupled with glutathione-Sepharose beads for 60 min at 4 °C. The beads were washed three times with CHAPS buffer and bound Raptor, Rictor or mTOR were detected by SDS-PAGE followed by Western blot using corresponding antibody. Immunoprecipitation HEK293T cells were washed with PBS, suspended with CHAPS buffer. Cell lysates were precleared and supernatant fluid was incubated with 20µl of monoclonal anti-Flag agarose beads for 1hour at 4℃. Immunoprecipitates were washed three times with CHAPS buffer and bound proteins were detected by SDS-PAGE followed by Western blot. Plasmids The FilGAP siRNA KD#2-resistant construct (rKD#2) was generated by introducing point mutations at nucleotide positions 771, 777, 780, 786, and 792 of the FilGAP coding sequence using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Sin1 was cloned from HEK293T cells by RT-PCR, and subcloned into pCMV5-Flag, pCMV5-Myc and pMAL-c2X. FilGAP, Raptor and Sin1 deletion mutants were generated by PCR. pEF-Bos-Flag-Rictor, pRK5Myc-Raptor was gifted from T Sato (Aichi Cancer Research Institute). RT-PCR cDNA was synthesized from 0.5 mg of total RNA by ReverTraAce (TOYOBO) and amplification was carried out using specific forward primers for ARHGAP24 gene as follows 45 : variant 1 primer located: 5’- CTG CAA TGA AGA GAA CCC AG-3’, variant 2: 5’- ATG CCT GAA GAC CGG AAT TC-3’, variant 3: 5’- CAG TGG ACA GTT AAA CAA GAG-3’, and variant 4: 5’- GTCACTGACCACTGAAGTGT-3’. Common reverse primer located 5’-CAT AAC GAA CAG TAT CCT CCA G-3’. β-actin was used as a loading control. Spheroid culture For spheroid generation, 200 ml/well of control or FilGAP-depleted KINGS-1 and U-87MG cells suspensions at optimized densities (0.5×10⁴ cells/mL) were dispensed into 96-well round-bottomed ultra-low attachment surface plates (Corning or Greiner). Spheroids were incubated for 9 days at 37°C, 5% CO₂ with 50% medium exchange every 3 days. Images of the spheroids were acquired every 3 days and their diameters were measured with Image J. Declarations DataAvailability. The datasets generated and analysed, and full sets of results obtained during the current study are available from the corresponding author on reasonable request. Acknowledgment We thank Dr. Tatsuhiro Saito (Aichi cancer institute) for providing plasmids, antibodies, and helpful advice. Author Contributions K.T., A.N., T.T., M.M., Y.M., performed the analysis and experiments. K.T., and Y.O., designed the experiments. K.T., A.N., and Y.O., wrote the paper. All authors reviewed the manuscript. Competing Interests : The authors declare no competing interests. References Kim, D.-H. et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002). Hara, K. et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002). Sarbassov, D. D. et al. 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Nakahara, S., Tsutsumi, K. & Zuinen, T. FilGAP, a Rho–ROCK-regulated GAP for Rac, controls adherens junctions in MDCK cells. Journal of Cell (2015). Zuinen, T., Tsutsumi, K. & Ohta, Y. FilGAP regulates distinct stages of epithelial tubulogenesis. Biochem. Biophys. Res. Commun. 514, 742–749 (2019). Gould, R. A. et al. Cyclic Mechanical Loading Is Essential for Rac1-Mediated Elongation and Remodeling of the Embryonic Mitral Valve. Curr. Biol. 26, 27–37 (2016). Hara, A. et al. The role of FilGAP, a Rac-specific Rho-GTPase-activating protein, in tumor progression and behavior of astrocytomas. Cancer Med. 5, 3412–3425 (2016). Feng, M. et al. RASAL2 activates RAC1 to promote triple-negative breast cancer progression. J. Clin. Invest. 124, 5291–5304 (2014). Yang, W. et al. ARHGAP24 represses β-catenin transactivation-induced invasiveness in hepatocellular carcinoma mainly by acting as a GTPase-independent scaffold. Theranostics 12, 6189–6206 (2022). Nishi, T. et al. FilGAP, a Rac-specific Rho GTPase-activating protein, is a novel prognostic factor for follicular lymphoma. Cancer Med. 4, 808–818 (2015). Tsutsumi, K. et al. AGAP1 regulates subcellular localization of FilGAP and control cancer cell invasion. Biochem. Biophys. Res. Commun. 522, 676–683 (2020). Saito, K., Ozawa, Y., Hibino, K. & Ohta, Y. FilGAP, a Rho/Rho-associated protein kinase-regulated GTPase-activating protein for Rac, controls tumor cell migration. Mol. Biol. Cell 23, 4739–4750 (2012). Saci, A., Cantley, L. C. & Carpenter, C. L. Rac1 regulates the activity of mTORC1 and mTORC2 and controls cellular size. Mol. Cell 42, 50–61 (2011). Hernández-Negrete, I. et al. P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration. J. Biol. Chem. 282, 23708–23715 (2007). Kawaguchi, K., Saito, K., Asami, H. & Ohta, Y. ADP ribosylation factor 6 (Arf6) acts through FilGAP protein to down-regulate Rac protein and regulates plasma membrane blebbing. J. Biol. Chem. 289, 9675–9682 (2014). Nguyen, L. S. et al. Transcriptome profiling of UPF3B/NMD-deficient lymphoblastoid cells from patients with various forms of intellectual disability. Mol. Psychiatry 17, 1103–1115 (2012). Su, Z.-J. et al. A vascular cell-restricted RhoGAP, p73RhoGAP, is a key regulator of angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 101, 12212–12217 (2004). Ohgaki, H. & Kleihues, P. Genetic pathways to primary and secondary glioblastoma. Am. J. Pathol. 170, 1445–1453 (2007). Fu, W. & Hall, M. N. Regulation of mTORC2 Signaling. Genes 11, (2020). Xie, J. & Proud, C. G. Crosstalk between mTOR complexes. Nature cell biology vol. 15 1263–1265 (2013). Chen, C.-Y., Chen, J., He, L. & Stiles, B. L. PTEN: Tumor Suppressor and Metabolic Regulator. Front. Endocrinol. 9, 338 (2018). Li, J. et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943–1947 (1997). Brady, S. W., Zhang, J., Tsai, M.-H. & Yu, D. 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Supplementary Files SupplementaryInfo.pdf supplementallytable.xlsx Cite Share Download PDF Status: Published Journal Publication published 08 Dec, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 13 Oct, 2023 Reviews received at journal 04 Oct, 2023 Reviewers agreed at journal 22 Sep, 2023 Reviews received at journal 22 Aug, 2023 Reviewers agreed at journal 09 Aug, 2023 Reviewers agreed at journal 09 Aug, 2023 Reviewers invited by journal 07 Aug, 2023 Editor assigned by journal 07 Aug, 2023 Editor invited by journal 07 Aug, 2023 Submission checks completed at journal 07 Aug, 2023 First submitted to journal 31 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3219907","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":224598107,"identity":"a0ec0224-f8c0-4253-b1b7-09202e87e0e1","order_by":0,"name":"Koji Tsutsumi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBACCSjNw8/D2IAiw4xbC1DqAFCLZA9jYwOSGYS1MBicYWBE0YITSDbwH5P+2LZNxvjM4fYHP/7Y1fEz8Bgw/KhhYDfHoUWagZlN4mDbbR6zs42Njb1tyRKSDTwGjD3HGJgtG7BrkYNrOQ/0C28Ds4TB/TcGDLwNDMwGBwhoMe5nbGz886dewuAA0Ja/eLTAHWbA29jYzMN2GKyFGZ8tks3MxhZnzt3mkThzsHG2bNtxyZkNbAWHZY5J4PSLxPHGhzcqym7b8/ekP/j45k81Pz8D88aHb2psknGFGPYYAzpJItkAlxacwI50LaNgFIyCUTBMAQD5i1HPfU+jwwAAAABJRU5ErkJggg==","orcid":"","institution":"Kitasato University","correspondingAuthor":true,"prefix":"","firstName":"Koji","middleName":"","lastName":"Tsutsumi","suffix":""},{"id":224598108,"identity":"5ffd9073-e204-4474-84e7-6782675a1f2f","order_by":1,"name":"Ayumi Nohara","email":"","orcid":"","institution":"Kitasato University","correspondingAuthor":false,"prefix":"","firstName":"Ayumi","middleName":"","lastName":"Nohara","suffix":""},{"id":224598109,"identity":"2bd7f331-ec55-40a7-a2f6-92c3e0570892","order_by":2,"name":"Taiki Tanaka","email":"","orcid":"","institution":"Kitasato University","correspondingAuthor":false,"prefix":"","firstName":"Taiki","middleName":"","lastName":"Tanaka","suffix":""},{"id":224598110,"identity":"cc51bdd6-a1cf-4526-870a-3f740dea3e9a","order_by":3,"name":"Moe Murano","email":"","orcid":"","institution":"Kitasato University","correspondingAuthor":false,"prefix":"","firstName":"Moe","middleName":"","lastName":"Murano","suffix":""},{"id":224598111,"identity":"45d7f401-1be9-4913-95d9-8d27de5c1f03","order_by":4,"name":"Yurina Miyagaki","email":"","orcid":"","institution":"Kitasato University","correspondingAuthor":false,"prefix":"","firstName":"Yurina","middleName":"","lastName":"Miyagaki","suffix":""},{"id":224598112,"identity":"90cdf0c8-0f7b-46d3-aead-1da5bfc56f31","order_by":5,"name":"Yasutaka Ohta","email":"","orcid":"","institution":"Kitasato University","correspondingAuthor":false,"prefix":"","firstName":"Yasutaka","middleName":"","lastName":"Ohta","suffix":""}],"badges":[],"createdAt":"2023-07-31 09:00:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3219907/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3219907/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-47892-1","type":"published","date":"2023-12-08T15:01:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":41438622,"identity":"d4be0035-0766-430c-a3dc-9aa400004f2f","added_by":"auto","created_at":"2023-08-11 14:01:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":612256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilGAP interacts with mTORC1/2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HEK293T cells were transfected with Flag-FilGAP and Myc-Raptor. After precipitation with Flag antibody, coprecipitated Raptor was detected with anti-Raptor antibody. (B) HEK293T cells were transfected with Flag-FilGAP (WT or V734Y) and Myc-Rictor. After precipitation with HA antibody, coprecipitated Rictor was detected with anti-Rictor antibody. (C) The purified recombinant GST-FilGAP (373-748aa) or GST alone were coupled to glutathione sepharose, and incubated with HEK293T cells lysed in buffer containing CHAPS. The washed precipitates were immunoblotted for the presence of Raptor, Rictor and mTOR. (D) GST-FilGAP (373-748aa) coupled to glutathione sepharose were incubated with HEK293T cells lysed in buffers containing CHAPS or Triton X-100. The washed precipitates were immunoblotted for the presence of Raptor, Rictor and mTOR. (E) siRNAs against Raptor or Rictor were transfected HEK293T cells. 72h later, cells were lysed in buffer containing CHAPS. Cell lysates were incubated with GST-FilGAP coupled to glutathione sepharose, and the washed precipitates were immunoblotted for the presence of Raptor, Rictor and mTOR. (F) HEK293T cells were treated with 100 nM Rapamycin or 500 nM Torin-1 for 1 hour. Cell lysates were subjected to GST pull down assay with GST-FilGAP 373-748aa. (G) The amount of Raptor, Rictor, and mTOR precipitated with FilGAP in (F) was calculated and presented as the mean ±SE. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 (Student’s t-test).\u003c/p\u003e","description":"","filename":"fIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/f1f08b14c368c9970a13f1c6.png"},{"id":41438616,"identity":"8e2462c5-e259-41fe-bae4-96587f9a1838","added_by":"auto","created_at":"2023-08-11 14:01:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1052218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of FilGAP and mTORC1/2 association region\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of FilGAP deletion mutants used to examine the interaction with Raptor. (B) Cell lysates prepared from Myc-Raptor transfected HEK293T cells were incubated with GST-FilGAP bound beads. Coprecipitated Myc-Raptor and mTOR were detected by immunoblotting. (C) Schematic diagram of FilGAP deletion mutants used to examine the interaction with Rictor. The red dotted line is the estimated Rictor binding region. (D) Cell lysates prepared from Flag-Rcitor transfected HEK293T cells were incubated with GST-FilGAP bound beads. Coprecipitated Flag-Rictor was detected by immunoblotting. (E) Schematic diagram of Raptor deletion mutants. (F) Raptor NT and CT was coprecipitated with GST-FilGAP 373-748aa. (G) Flag-Rictor or Flag-Sin1 was transfected to HEK293T cells and cell lysates were subjected to pull down assay using GST-FilGAP 455-600aa. (H) Schematic diagram of Sin1 deletion mutants. (I) Purified MBP-Sin1 Full and NT was pulled down by GST-FilGAP 455-600aa.\u003c/p\u003e","description":"","filename":"fIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/dc226e026c6045d6e70c0d99.png"},{"id":41438618,"identity":"764d5889-a4f4-49f4-9f01-4d093a75ed11","added_by":"auto","created_at":"2023-08-11 14:01:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":205082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilGAP activates mTORC1/2 in glioma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Human astrocytoma KINGS-1 cells were transfected with siRNAs against FilGAP. Immunoblots for P-S6K and P-AKT represent the activities of mTORC1 and mTORC2 respectively. Actin was the loading control. (B) P-S6K/Total S6K and P-AKT/Total AKT ratio in (A) were calculated (n=6). *P\u0026lt;0.05, **P\u0026lt;0.01 (ANOVA Tukey HDS Test). (C) control or FilGAP-depleted KINGS-1 cells were transfected with HA-FilGAP resistant to FilGAP siRNA KD#2 (FilGAP rKD#2). Tubulin was the loading control. (D) P-S6K/Total S6K and P-AKT/Total AKT ratio in (C) were calculated (n=5). *P\u0026lt;0.05, **P\u0026lt;0.01 (Student’s t-test).\u003c/p\u003e","description":"","filename":"fIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/3d9efb25d59589c5a4508a48.png"},{"id":41440304,"identity":"3e8a17c2-735b-4d19-b21e-9dc66aaff26d","added_by":"auto","created_at":"2023-08-11 14:09:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":168169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePH domain of FilGAP is important for regulation of mTORC2 activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of FilGAP constructs. (B) KINGS-1 cells were transfected with HA-FilGAP rKD#2 constructs (WT, R175A or ΔPH) after depletion of FilGAP with siRNA. (C, D) P-AKT/Total AKT and P-S6K/Total S6K ratio in (B) were calculated (n=6). *P\u0026lt;0.05, **P\u0026lt;0.01 (Student’s t-test).\u003c/p\u003e","description":"","filename":"fIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/73f5e684ed2f7d1485f28096.png"},{"id":41442984,"identity":"fd6d37a0-db78-42e8-8915-b9b9af8ef9ea","added_by":"auto","created_at":"2023-08-11 14:17:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":372116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilGAP partial PH-deleted variant activates mTORC1/2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of FilGAP variants expressed in KINGS-1 cells. (B) RT-PCR analysis of expression of FilGAP variants in A7 melanoma, MDA-MB-231 breast cancer, U87MG glioma and KINGS-1 glioma cell line. β-actin was used as a loading control. (C) KINGS-1 cells were transfected with siRNA targeted against all FilGAP variants (FilGAP KD#1) or only variant 1. (D) P-S6K/Total S6K and P-AKT/Total AKT ratio in (B) were calculated (n=3). (E) KINGS-1 cells were transfected with HA-FilGAP rKD#1 constructs (WT, 96-748aa) after depletion of FilGAP with siRNA. (F) P-S6K/Total S6K and P-AKT/Total AKT ratio in (E) were calculated (n=8). *P\u0026lt;0.05, **P\u0026lt;0.01 (Student’s t-test). (G) HEK293T cells were transfected with HA-FilGAP (WT or 96-748aa) and Flag-Rictor. 24h after transfection, cells were lysed and after immunoprecipitation with anti-FilGAP antibody.\u003c/p\u003e","description":"","filename":"fIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/93accd371dc0bffafaedcb5a.png"},{"id":41444560,"identity":"d70539cd-a2f0-4435-bed6-f32e428b241b","added_by":"auto","created_at":"2023-08-11 14:25:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":333969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilGAP is involved in PI3K-independent mTORC2 activation in glioblastoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunoblots for FilGAP expression and mTORC1/2 activities in astrocytoma KINGS-1 and glioblastoma U-87MG cells. (B) P-S6K/Total S6K, P-AKT/Total AKT ratio and amount of FilGAP expression in (A) were calculated (n=3). (C) U-87MG cells were transfected with siRNAs against FilGAP. (D) P-S6K/Total S6K and P-AKT/Total AKT ratio in (C) were calculated (n=3). (E) Control or FilGAP-depleted U-87MG cells were treated with 10 mM LY294002 for 1 hour. (F) P-AKT/Total AKT ratio in (E) were calculated (n=5). *P\u0026lt;0.05, **P\u0026lt;0.01 (Student’s t-test).\u003c/p\u003e","description":"","filename":"fIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/4cd0e8b35147b06849a8edf9.png"},{"id":41440300,"identity":"8c255d8b-e73b-4247-b0e9-ae45c8eb7a98","added_by":"auto","created_at":"2023-08-11 14:09:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":398347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emTOR and FilGAP regulate spheroid growth in KINGS-1 and U-87MG cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of KINGS-1 and U-87MG spheroids cultured in a round-bottom low attachment 96 well plate, and treated with DMSO, 10 mM LY294002, 500 nM Torin1 or 100 nM Rapamycin for 12 days. Scale bar: 300 mm. (B) The diameter of each spheroid of (A) was measured (n=3). (C) Representative images of control or FilGAP-depleted KINGS-1 and U-87MG spheroids cultured for 9 days. Scale bar: 200 mm. (D) The diameter of each spheroid of (C) was measured (KINGS-1; n=3) (U-87MG; n=6). (E) Control or FilGAP-depleted KINGS-1 and U-87MG spheroids treated with DMSO, 10 μM LY294002, 500 nM Torin1 or 100 nM Rapamycin for 9 days. The diameter of each spheroid on day 9 of was measured (n=3).\u003c/p\u003e","description":"","filename":"fIG7.png","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/cfbaff84e2ef1c4c21085c2b.png"},{"id":47989259,"identity":"3f68e0e9-76b4-493f-abe8-bc663186cf2a","added_by":"auto","created_at":"2023-12-11 15:08:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2112833,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/fb99bfd9-2340-481b-b470-2187fc9ae2ee.pdf"},{"id":41438625,"identity":"316e9e9f-c94d-4654-8aee-3d2a8018261c","added_by":"auto","created_at":"2023-08-11 14:01:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5180031,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfo.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/7e8f71aa0fc7f87465de65f8.pdf"},{"id":41440302,"identity":"a3aaf19c-25d2-4144-8448-5e0aa2940d00","added_by":"auto","created_at":"2023-08-11 14:09:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11453,"visible":true,"origin":"","legend":"","description":"","filename":"supplementallytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3219907/v1/be680617084ca09a3be890d3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that is highly conserved from yeast to mammals belonging to the PI3K-related protein kinases family. mTOR forms functionally distinct protein complexes, mTORC1 and mTORC2. mTORC1 has Raptor, and mTORC2 has Rictor and Sin1 as specific subunits respectively \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. mTORC1 is activated by amino acids, and regulates protein synthesis and autophagy by phosphorylating downstream substrates, such as S6 kinase and 4EBP1 \u003csup\u003e5\u0026ndash;8\u003c/sup\u003e. mTORC2 is primarily activated by growth factors such as insulin, and regulates cell proliferation, cell survival, and cell migration by phosphorylating AKT, PKC and other substrates \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While the detailed mechanism of mTORC1 activation has been clarified, the molecular mechanism of mTORC2 activation remains unclear. Since new regulators of mTORC1 and mTORC2 activity have been reported even recently, the regulatory pathway of mTOR is considered to be very complex \u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. mTOR signaling is activated in a variety of tumors \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In particular, in glioma, one of the malignant brain tumors, activation of the mTOR pathway promotes cell proliferation and invasion, and contributes to patient poor prognosis \u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRho small GTPases cycle between active GTP-bound and inactive GDP-bound state, and act as key regulators of actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) activate Rho GTPase by catalyzing the exchange of GDP for GTP. While GTPase-activating proteins (GAPs) stimulate the intrinsic GTPase activity and inactivate them \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. FilGAP (also known as ARHGAP24) is a Filamin A-binding protein, and Rac-specific GAP \u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. FilGAP suppresses leading edge protrusion and promotes cell retraction by inhibiting Rac and also regulates epithelial cell-cell adhesion and tissue morphogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In addition, it has been suggested that the expression level of FilGAP is associated with malignant transformation of cancer and is also involved in invasive metastasis and tumorigenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38 CR39 CR40\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, the contribution of FilGAP to cancer development is thought to be different among cancer types, and more detailed mechanisms need to be elucidated.\u003c/p\u003e \u003cp\u003eIn this study, we found that FilGAP interacts with mTORC1 and mTORC2. We showed that Raptor, Rictor and mTOR were coprecipitated with FilGAP. Depletion of FilGAP expression in KINGS-1 gliomas had an inhibitory effect on S6K and AKT phosphorylation, while overexpression of FilGAP had a promotive effect, suggesting that FilGAP activates mTORC1 and mTORC2. A short FilGAP variant lacking partial PH domain was highly expressed in gliomas, and this short variant also contributed to mTORC1 and mTORC2 activation. Depletion of FilGAP did not affect mTOR activity in U-87MG, a highly malignant glioblastoma, but significantly decreased AKT phosphorylation in the presence of a PI3K inhibitor. Finally, we showed that depletion of FilGAP inhibited spheroid formations in KINGS-1 and U-87MG as well as mTOR inhibition. These results suggest that FilGAP interacts with mTORC1 and mTORC2 and may positively regulate their activity to promote tumorigenesis in gliomas.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFilGAP interacts with mTORC1 and mTORC2\u003c/h2\u003e \u003cp\u003eRecently, it has been reported that mTORC2 interacts with an actin filament crosslinking protein, Filamin A in glioblastoma cells \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, mTOR has been reported to interact with various Rho family GTPases and their regulators \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Thus, we considered the possibility that FilGAP interacts with mTORC2 through Filamin A. To examine whether FilGAP interacts with mTOR complex, we transfected Raptor or Rictor with or without FilGAP in HEK293T cells and performed coimmunoprecipitation assay (Fig.\u0026nbsp;1A, B). Both Raptor and Rictor were coprecipitated with FilGAP, suggesting that FilGAP interacts with Raptor and Rictor. Rictor was also coprecipitated with FilGAP V734Y, Filamin A-binding deficient mutant, suggesting that this interaction is independent of Filamin A. Further, we investigated whether mTOR components in HEK293T cell lysates were pulled down with GST-FilGAP (373-748aa) (Fig.\u0026nbsp;1C). mTOR, Raptor and Rictor were co-precipitated with GST-FilGAP. This result suggests, that FilGAP may interact with both mTORC1 and mTORC2. The mTOR complexes are disrupted in the buffer with non-ionic detergent such as TritonX-100, but can be maintained in the buffer containing amphoteric detergent CHAPS \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, we prepared cell lysates with TritonX-100 or CHAPS and performed GST-pulldown assay (Fig.\u0026nbsp;1D). In the presence of TritonX-100, the coprecipitation with GST-FilGAP was decreased for Raptor and mTOR, but slightly increased for Rictor. We further examined the interaction of mTOR complex with FilGAP in Raptor- or Rictor-depleted cells (Fig.\u0026nbsp;1E). Knockdown of Raptor, but not Rictor, reduced the binding of mTOR to FilGAP, suggesting that most of the mTOR coprecipitated with FilGAP is mediated by Raptor. We examined whether binding to FilGAP is altered when the kinase activity of the mTOR is suppressed. Treatment of the cells with Rapamycin, mTORC1 specific inhibitor, and Torin1, mTOR inhibitor significantly decreased the amount of Raptor and mTOR coprecipitated with FilGAP. On the other hand, Rictor coprecipitated with FilGAP was rather increased by the inhibitor treatments. Similar data were observed when mTORC1 was activated by amino acids and insulin stimulation. (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). These data suggest that the interaction of mTORC1 and FilGAP is dependent on mTORC1 activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of domains mediating FilGAP-mTORC1/2 interaction\u003c/h2\u003e \u003cp\u003eTo clarify the manner of the interaction between FilGAP and mTOR complexes, we examined detailed interaction between FilGAP and mTOR components. We generated a series of FilGAP-deletion mutants and examined their binding regions to Raptor or Rictor (Fig.\u0026nbsp;2A-D, Fig. S2A, B). C-terminus deletion resulted in a substantial decrease in interaction with Raptor, suggesting that the C-terminal region of FilGAP is important for binding to Raptor (Fig.\u0026nbsp;2A, B, Fig. S2A, B). On the other hand, it is suggested that FilGAP 500-520aa and 560-570aa are important for binding to Rictor (Fig.\u0026nbsp;2C, D). We generated deletion mutants of Raptor and examined their binding to GST-FilGAP and found that it binds to NT and CT but not to MD (Fig.\u0026nbsp;2E, F, Fig. S2C). Sin1, a specific component of mTORC2, also coprecipitated with FilGAP and the N-terminus of Sin1 was important for this binding. (Fig.\u0026nbsp;2G, H, Fig. S2D). Sin1 and FilGAP also co-precipitated in binding experiments with purified proteins, suggesting that this binding is direct (Fig.\u0026nbsp;2H).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFilGAP activates mTORC1/2 in glioma cells\u003c/h2\u003e \u003cp\u003eTo study the role of interaction between mTORC1/2 and FilGAP, we used KINGS-1 human astrocytoma cell line, which expresses high level of FilGAP \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Small interference RNAs (siRNAs) targeting FilGAP were transfected to KINGS-1 cells. Depletion of FilGAP in KINGS-1 cells significantly reduced phosphorylation of S6K and AKT, which are substrates of mTORC1 and mTORC2, respectively (Fig.\u0026nbsp;3A, B). Furthermore, transfection of HA-tagged FilGAP in KINGS-1 cells increased phosphorylation of S6K and AKT, and rescued the decrease in their phosphorylation caused by depletion of FilGAP (Fig.\u0026nbsp;3C, D). These results suggest that FilGAP may activate mTORC1/2 in KINGS-1 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePH domain of FilGAP is important for regulation of mTORC2 activity\u003c/h2\u003e \u003cp\u003eFilGAP contains pleckstrin homology (PH), Rho GAP, and coiled-coil (CC) domains \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. FilGAP R175A has mutation at GAP domain and lacks GAP activity. PH domain of FilGAP is required for Phosphatidylinositol 3-phosphate (PIP3)-dependent membrane localization \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. To study whether GAP activity and PH domain of FilGAP is required for the regulation of mTORC1/2 activities, HA-FilGAP constructs (WT, R175A or DPH; Fig.\u0026nbsp;4A) resistant to FilGAP siRNA were transfected to KINGS-1 cells after depletion of endogenous FilGAP with siRNA (Fig.\u0026nbsp;4B). Although FilGAP wild-type (WT) and R175A significantly increased AKT phosphorylation, FilGAP DPH did not increase AKT phosphorylation as much as WT and R175A (Fig.\u0026nbsp;4C). On the other hand, all FilGAP constructs increased S6K phosphorylation to the same extent (Fig.\u0026nbsp;4D). These results suggest that PH domain of FilGAP is important for the regulation of mTORC2 activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFilGAP partial PH-deleted transcript variant activates mTORC1/2\u003c/h2\u003e \u003cp\u003eThere are multiple FilGAP variants, some of which are partially or completely lacking PH domain (Fig.\u0026nbsp;5A) \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The expression of FilGAP variants was examined by RT-PCR analysis and variants 3 and 4 were highly expressed in gliomas (U-87MG and KINGS-1) compared to melanoma (A7) and breast cancer cells (MDA-MB-231) (Fig.\u0026nbsp;5B). To study whether FilGAP variants 3 and 4 also regulate mTOR activity, siRNA targeting specific FilGAP variant 1 was transfected to KINGS-1 cells. Knockdown of FilGAP variant 1\u0026ndash;4 (all isoforms) and only FilGAP variant 1 were confirmed by immunoblotting (Fig.\u0026nbsp;5B). Depletion of all FilGAP variants by siRNA KD#1 decreased phosphorylation of S6K and AKT, while depletion of only FilGAP variant 1 significantly increased their phosphorylation. (Fig.\u0026nbsp;5B, C). In addition, depletion of only FilGAP variant 1 tended to increase the expression levels of variants 3 and 4. To further investigate the effect of FilGAP variant 3 on the activities of mTORC1/2, HA-FilGAP constructs (96-748aa; variant 3) resistant to FilGAP siRNA was transfected to KINGS-1 cells after depletion of FilGAP with siRNA. FilGAP (96-748aa) increased mTORC1/2 activities to the same extent as WT (Fig.\u0026nbsp;5D, E). Interestingly, FilGAP 96\u0026ndash;748 aa showed increased coimmunoprecipitation with Rictor rather than WT (Fig.\u0026nbsp;5F). These results suggest that FilGAP variant 3, 4 may have a higher or equal ability to activate mTORC1/2 compared to variant 1. Give that ΔPH (131-748aa) cannot increased mTORC2 activity, 96\u0026ndash;130 aa of FilGAP may be important to activate mTORC2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFilGAP is involved in PI3K-independent mTORC2 activation in glioblastoma cells\u003c/h2\u003e \u003cp\u003eGliomas are classified from grade II to grade IV according to histological and genetic diagnosis \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Grade IV glioblastoma is the most aggressive and has a very poor prognosis with a 2-year survival rate of less than 30%. To investigate the relationship between the mTORC1/2 regulation by FilGAP and grade of glioma malignancy, we compared mTORC1/2 activities and the expression levels of FilGAP in KINGS-1 and U-87MG, grade III glioma and grade IV glioblastoma, respectively. Phosphorylation of S6K and AKT were higher in U-87MG than in KINGS-1, suggesting that mTORC1 and mTORC2 were more active in U-87MG. (Fig.\u0026nbsp;6A). The expression levels of FilGAP was higher in KINGS-1 cells than in U-87MG (Fig.\u0026nbsp;6B). To study whether FilGAP regulates mTORC1/2 activities in U-87MG cells as observed in KINGS-1, siRNAs targeting FilGAP was transfected to U-87MG and examined phosphorylation of AKT and S6K. Depletion of FilGAP in U-87MG cells did not affect phosphorylation of S6K and AKT, unlike in KINGS-1 cells (Fig.\u0026nbsp;6C, D). Since mTORC1/2 activities are very high in U87MG, we postulated that the contribution of FilGAP to mTORC1/2 activities may be small. PI3K signaling, one of the upstream activator of mTOR, is known to be activated in glioblastoma by genetic mutations or deletions. So, we examined the effect of FilGAP depletion in U-87MG cells in the presence of PI3K inhibitor (LY294002). PI3K inhibition greatly reduced phosphorylation of AKT and almost completely abolished phosphorylation of S6K. And depletion of FilGAP significantly decreased AKT phosphorylation in U-87MG cells in the presence of PI3K inhibitor (Fig.\u0026nbsp;6E, F). These results suggest that FilGAP may be involved in PI3K-independent mTORC2 activation in glioblastoma cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFilGAP regulate spheroid growth in KINGS-1 and U-87MG cells\u003c/h2\u003e \u003cp\u003emTOR regulates glioma survival, proliferation, and invasion. To study the role of mTORC1/2 regulation by FilGAP in glioma, we examined whether inhibition of mTOR or depletion of FilGAP affect tumorigenesis using 3D spheroid model of KINGS-1 and U-87MG cells. When KINGS-1 and U-87MG were seeded on low-attachment U-bottom plates, the spheroid size increased with the number of days in culture. We used PI3K inhibitor (LY294002), mTOR inhibitor (Torin1), and mTORC1 inhibitor (Rapamycin) to inhibit mTOR activity. All inhibitors treatment reduced spheroid diameter in both KINGS-1 and U-87MG cells, indicating that mTOR activity is critical for spheroid growth (Fig.\u0026nbsp;7A, B). Depletion of FilGAP also reduced spheroid diameter in both KINGS-1 and U-87MG (Fig.\u0026nbsp;7C, D), suggesting that FilGAP promotes spheroid growth in glioma as well as mTOR. Finally, we examined the effects of a combination of mTOR inhibition and suppression of FilGAP expression on spheroid growth. The combination of mTOR inhibitor treatment and suppression of FilGAP expression significantly reduced spheroid size compared to either treatment alone (Fig.\u0026nbsp;7E, Fig. S3). These data suggest that the combination of mTOR inhibitor treatment and suppression of FilGAP expression has a potent inhibitory effect on tumorigenesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found that FilGAP interacts with mTORC1/2. FilGAP seems to interact with mTORC1 through Raptor and with mTORC2 through Rictor or Sin1 (Fig.1 and 2). We also found that FilGAP regulates mTORC1/2 activities in glioma cells. \u0026nbsp;PH domain (1-130aa) of FilGAP, especially the region 96-130aa, is important for mTORC2 activation (Fig. 6). In addition, we showed that FilGAP may be involved in PI3K-independent mTORC2 activation in glioblastoma. Finally, we showed that the suppression of FilGAP expression and mTOR inhibition attenuated spheroids growth in both KINGS-1 and U-87MG. Taken together, these results suggest that FilGAP may regulate tumor growth in gliomas through the regulation of mTORC1/2 activities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe showed that FilGAP interacts with mTORC1/2 and regulates their activities. However, it is still unclear how FilGAP regulates mTORC1/2 activities. mTORC1 is activated by amino acids and other nutrients though the Rheb and Rag GTPases\u003csup\u003e4\u003c/sup\u003e. On the other hand, mTORC2 is activated primarily by growth factors through PI3K signaling\u0026nbsp;\u003csup\u003e48\u003c/sup\u003e. One possibility is that FilGAP may promote complex formation of mTORC1 and mTORC2. In addition, there is a crosstalk pathway between mTORC1 and mTORC2 that regulates each other\u0026apos;s activity\u0026nbsp;\u003csup\u003e49\u003c/sup\u003e. Since FilGAP binds to both mTORC1 and mTORC2, it may be involved in the regulation of mTORC1 and mTORC2 cross-talk. Future clarification of the detailed pathway of mTOR activation by FilGAP may shed light on the very complex regulatory mechanism of mTOR pathway.\u003c/p\u003e\n\u003cp\u003eWe showed that FilGAP transcript variants partially lacking PH domain, which are highly expressed in gliomas, also activate mTORC1 and mTORC2 (Fig. 5). Further 96-130aa of FilGAP may be important for mTORC2 activation (Fig. 4, 5). FilGAP 96-748aa corresponding to variant 3 was suggested to bind Rictor more strongly than the full length (Fig. 5G). \u0026nbsp;It is possible that FilGAP 1-95aa acts to interfere with the interaction with Rictor. FilGAP with PH domain can also activate mTORC2, it is possible that the binding of PIP3 to the PH domain of FilGAP affects its interaction with mTORC2. Since FilGAP 1-95aa contains Arginine 39, which is important for binding to PIP3, it is considered that FilGAP variant 3 cannot bind to PIP3\u0026nbsp;\u003csup\u003e44\u003c/sup\u003e. Therefore, it is likely that FilGAP lacking PH domain can interact with mTORC2 in a PIP3-independent manner.\u003c/p\u003e\n\u003cp\u003eDepletion of FilGAP in U-87MG, grade IV glioblastoma cell line, did not affect mTORC1/2 activities (Fig. 6). Since highly malignant glioma such as glioblastoma acquires high mTORC1/2 activities by the gene mutations, the contribution of FilGAP to mTORC1/2 activities seems to be reduced. Actually, U-87MG cells have mutations in PTEN, a negative regulator of mTOR pathway\u0026nbsp;\u003csup\u003e50,51\u003c/sup\u003e. However, depletion of FilGAP in U-87MG decreased mTORC2 activity in the presence of PI3K inhibitors (Fig. 6E, F). This suggests that FilGAP may regulate mTORC2 activity PI3K-independent manner. PI3K-independent activation of mTOR has been reported in drug-resistant cancer cells\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e. FilGAP may be involved in the acquisition of drug resistance by cancer cells with high PI3K activity.\u0026nbsp;Remarkably, glioma cells express high levels of FilGAP variant 3 and 4, which structurally appears to function in a PIP3-independent manner. FilGAP transcript variant lacking a part of PH domain may be important for PI3K-independent survival of cancer cells. This may be the reason that depletion of FilGAP suppresses spheroid growth not in KINGS-1 cells but also U-87MG cells (Fig. 7C, D). Since the low adhesion and hypoxic conditions during spheroid formation affect the PI3K pathway\u0026nbsp;\u003csup\u003e53\u003c/sup\u003e, the regulation of mTORC1/2 activities by FilGAP might be prominently observed.\u003c/p\u003e\n\u003cp\u003eThis study provides FilGAP as a new PI3K-independent mTORC1/2 activator. FilGAP may become a potential therapeutic target for drug-resistant glioblastoma. It will be important issue to be solved to determine how FilGAP activates mTORC1/2, and the role of this regulation \u003cem\u003ein vivo.\u003c/em\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture, reagents and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKINGS-1 cells (GIII astrocytoma, HSRRB) were maintained in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin and 50 mg/ml streptomycin. U-87MG (Glioblastoma, ATCC) cells were maintained in Eagle\u0026apos;s minimal essential medium (E-MEM) supplemented with 10% FBS, 50 units/ml penicillin and 50 mg/ml streptomycin. HEK293T cells, MDA-MB-231cells (ATCC) were maintained in\u0026nbsp;Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% FBS.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eA7 cells were provided from Dr. Thomas P. Stossel (Harvard University). A7 cells were maintained in Minimum Essential Medium Eagle supplemented with 8% newborn calf serum, 2% fetal calf serum, 50 units/ml penicillin and 50 mg /ml streptomycin. Reagents, antibodies, siRNA used in this study were listed in supplementally table 1. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKINGS-1 cells were transfected with plasmid DNA for 24 hours using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u0026apos;s instructions. HEK293T cells was transfected with plasmid DNA for 24 hours using Poly-Ethylene-Imine (PEI, polyscience). KINGS-1, U-87MG cells were transfected with FilGAP siRNA for 48 or 72 hours using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. The cells were maintained at 37\u0026deg;C with 5% CO₂ during the treatments. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal cellular proteins were harvested using RIPA buffer [20 mM Tris‐HCl (pH7.5), 120 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EDTA, 10 mM NaF, 10 mM b-glycerophosphate, Protease inhibitor Cocktail and 1 mM DTT]. Cell lysates were separated by SDS‐PAGE and transferred to the membrane for fluorescence (Millipore). The membrane was blocked with Intercept (PBS) Blocking Buffer (LI-COR) and incubated with primary antibodies. The primary antibodies were detected with secondary antibodies and FUSION SOLO S (Vilber Loumat).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGST pulldown assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T cells were washed with phosphate-buffered saline (PBS), suspended with CHAPS buffer (50mM Tris-HCl [pH 7.5], 0.1M NaCl, 2mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1mM EDTA [pH 8.0], 0.3% CHAPS, Protease inhibitor Cocktail (SIGMA), and 1mM DTT). The cells were disrupted and the cell lysates were prepared by centrifugation for at 15,000 rpm for 5 min at 4\u0026deg;C. The supernatant fluid was incubated with GST-FilGAP protein coupled with glutathione-Sepharose beads for 60 min at 4 \u0026deg;C. The beads were washed three times with CHAPS buffer and bound Raptor, Rictor or mTOR were detected by SDS-PAGE followed by Western blot using corresponding antibody.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T cells were washed with PBS, suspended with CHAPS buffer. Cell lysates were precleared and supernatant fluid was incubated with 20\u0026micro;l of monoclonal anti-Flag agarose beads for 1hour at 4℃. Immunoprecipitates were washed three times with CHAPS buffer and bound proteins were detected by SDS-PAGE followed by Western blot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FilGAP siRNA KD#2-resistant construct (rKD#2) was generated by introducing point mutations at nucleotide positions 771, 777, 780, 786, and 792 of the FilGAP coding sequence using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Sin1 was cloned from HEK293T cells by RT-PCR, and subcloned into pCMV5-Flag, pCMV5-Myc and pMAL-c2X. FilGAP, Raptor and Sin1 deletion mutants were generated by PCR. pEF-Bos-Flag-Rictor, pRK5Myc-Raptor was gifted from T Sato (Aichi Cancer Research Institute). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecDNA was synthesized from 0.5 mg of total RNA by ReverTraAce (TOYOBO) and amplification was carried out using specific forward primers for \u003cem\u003eARHGAP24\u003c/em\u003e gene as follows \u003csup\u003e45\u003c/sup\u003e: variant 1 primer located: 5\u0026rsquo;- CTG CAA TGA AGA GAA CCC AG-3\u0026rsquo;, variant 2: 5\u0026rsquo;- ATG CCT GAA GAC CGG AAT TC-3\u0026rsquo;, variant 3: 5\u0026rsquo;- CAG TGG ACA GTT AAA CAA GAG-3\u0026rsquo;, and variant 4: 5\u0026rsquo;- GTCACTGACCACTGAAGTGT-3\u0026rsquo;. Common reverse primer located 5\u0026rsquo;-CAT AAC GAA CAG TAT CCT CCA G-3\u0026rsquo;. \u0026beta;-actin was used as a loading control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpheroid culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor spheroid generation, 200 ml/well of control or FilGAP-depleted KINGS-1 and U-87MG cells suspensions at optimized densities (0.5\u0026times;10⁴ cells/mL) were dispensed into 96-well round-bottomed ultra-low attachment surface plates (Corning or Greiner). Spheroids were incubated for 9 days at 37\u0026deg;C, 5% CO₂ with 50% medium exchange every 3 days. Images of the spheroids were acquired every 3 days and their diameters were measured with Image J.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDataAvailability.\u003c/strong\u003e The datasets generated and analysed, and full sets of results obtained during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Tatsuhiro Saito (Aichi cancer institute) for providing plasmids, antibodies, and helpful advice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.T., A.N., T.T., M.M., Y.M., performed the analysis and experiments. K.T., and Y.O., designed the experiments. K.T., A.N., and Y.O., wrote the paper. All authors reviewed the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKim, D.-H. \u003cem\u003eet al.\u003c/em\u003e mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163\u0026ndash;175 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHara, K. \u003cem\u003eet al.\u003c/em\u003e Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177\u0026ndash;189 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarbassov, D. D. \u003cem\u003eet al.\u003c/em\u003e Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. 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Cell Sci. 130, 203\u0026ndash;218 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3219907/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3219907/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that forms the two different protein complexes, known as mTORC1 and mTORC2. mTOR signaling is activated in a variety of tumors, including glioma that is one of the malignant brain tumors. FilGAP (ARHGAP24) is a negative regulator of Rac, a member of Rho family small GTPases. In this study, we found that FilGAP interacts with mTORC1/2 and is involved in tumor formation in glioma. FilGAP interacted with mTORC1 via Raptor and with mTORC2 via Rictor and Sin1. Depletion of FilGAP in KINGS-1 glioma cells decreased phosphorylation of S6K and AKT. Furthermore, overexpression of FilGAP increased phosphorylation of S6K and AKT, suggesting that FilGAP activates mTORC1/2. U-87MG, glioblastoma cells, showed higher mTOR activity than KINGS-1, and phosphorylation of S6K and AKT was not affected by suppression of FilGAP expression. However, in the presence of PI3K inhibitors, phosphorylation of S6K and AKT was also decreased in U-87MG by depletion of FilGAP, suggesting that FilGAP activates mTORC2 in PI3K-independent manner. Finally, we showed that depletion of FilGAP in KINGS-1 and U-87MG cells significantly reduced spheroid growth. These results suggest that FilGAP may contribute to tumor growth in glioma by regulating mTORC1/2 activities.\u003c/p\u003e","manuscriptTitle":"FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-11 14:01:26","doi":"10.21203/rs.3.rs-3219907/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-10-13T14:05:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-10-04T13:43:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6ff1449a-3b6d-479b-a910-9ba351d63c27","date":"2023-09-22T15:57:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-08-22T19:31:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7c492a79-f095-496b-9f3c-e2699539ceb8","date":"2023-08-09T17:39:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c2e8332a-880e-4d16-9db8-60cc1043a471","date":"2023-08-09T16:12:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-08-07T13:52:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-07T13:26:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-08-07T11:27:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-07T11:11:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-07-31T08:46:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f0bf85d-d7d3-4a99-b1e7-56591192bc0b","owner":[],"postedDate":"August 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":23857862,"name":"Biological sciences/Cell biology/Cell growth/Tor signalling"},{"id":23857863,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2023-12-11T15:05:27+00:00","versionOfRecord":{"articleIdentity":"rs-3219907","link":"https://doi.org/10.1038/s41598-023-47892-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-12-08 15:01:57","publishedOnDateReadable":"December 8th, 2023"},"versionCreatedAt":"2023-08-11 14:01:26","video":"","vorDoi":"10.1038/s41598-023-47892-1","vorDoiUrl":"https://doi.org/10.1038/s41598-023-47892-1","workflowStages":[]},"version":"v1","identity":"rs-3219907","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3219907","identity":"rs-3219907","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}