Background
Glioblastoma multiforme (GBM) is classified as a grade IV type for malignant gliomas according to the World Health Organization (WHO) system [
1]. It is one of the most lethal tumors based on survival and recurrence rates after diagnosis and resistant to both chemo- and radiotherapeutics. In the past, most cases after initial treatments of GBM such as surgery, radio- and chemotherapy are occasionally followed by recurring tumors which eventually progress to death [
2]. The epidermal growth factor receptor (EGFR) is one of the key contributors that contribute to cancer hallmarks in GBM [
3]. EGFR activating mutations are commonly observed in GBM patients and the EGFR variant III is the most common type of mutation conferring constitutive signals to many pathways downstream, mainly PI3K-AKT-mTOR, and MAPK [
4‐
8].
In brain cells, the mechanistic target of rapamycin complex 2 (mTORC2) has been implicated as one of the main regulators. Abnormality of mTORC2 signaling pathway is linked to malignant glioma and has been broadly studied [
9‐
12]. Several mutations, ablations, deregulated signaling pathways including mTORC2 are found to be key characteristics of malignant glioma [
13]. Also, mTORC2 is believed to maintain neuron morphology and synapse function which is lacking or deregulated in brain diseases [
14]. Furthermore, hyperactivated mTORC2 with RICTOR overexpression was evidenced in various types of brain cancers. Masri
et al. [
15] showed that elevated mTORC2 activity promotes tumorigenesis, tumor growth and proliferation. Levels of RICTOR protein and mTORC2 activity can be related to specific stages of cancer invasiveness [
15,
16]. The mTORC2 has been linked to metabolic reprogramming in GBM including glycolytic metabolism, glutaminolysis, lipogenesis, and nucleotide and ROS metabolism [
17]. These activations of several pathways by mTORC2 are believed to cause resistance to signaling inhibitors [
18]. Recently, TORC2 in yeast and mTORC2 have also been reported to be new important players in DNA damage control and genome stability. These processes are essential for survival of cancer cells during stress-mediated DNA damage and also for cancer development [
19‐
22].
The mTOR complex 2 (mTORC2) consists of the four main components which are mTOR, RICTOR, mSIN1 and mLST8 (GβL), and including other closely associated proteins such as PROTOR1/2, DEPTOR, TTI1 and TEL2 [
23]. mTOR is a serine-threonine protein kinase that belongs to the phosphoinositide-3-kinase-related kinase family [
24]. The complex is stimulated by growth factors critical for AKT activation resulting in the phosphorylation of AKT residue Ser473 [
25]. Activated AKT further signals downstream to contribute to events such as cell survival, apoptosis, lipid and glucose metabolisms, and via mTORC1 to promote cell proliferation, and protein synthesis [
26,
27]. On the other hand, mTORC2 has also been shown to control actin cytoskeleton reorganization independent of AKT [
28,
29]. One mechanism is by phosphorylating the hydrophobic motif of SGK1 and several isoforms of PKCs [
13,
30,
31] that are important for transcription regulation, actin cytoskeleton reorganization [
17]. Moreover, mTORC2-mediating actin polymerization is required for the regulation of both long-term memory formation and long-term synaptic plasticity in mice [
32]. Depletion of mTOR or RICTOR but not RAPTOR was recently demonstrated to impair migration, invasion, and stress fiber formation of highly migratory
Tsc2
-/-
mouse embryonic fibroblast cells [
33]. However, mechanisms that mTORC2 use to regulate morphology and motility of cells are still poorly understood.
To gain further insight into the function of mTORC2, we first examined effects of inhibiting mTORC2 by using the mTOR kinase inhibitor PP242 [
34]. This revealed alteration of cellular cytoskeleton as well as focal adhesions and inhibition of motility and invasion. Affinity purification and characterization of mTORC2 from GBM cells were performed and these led to the finding that Filamin A (FLNA) is associated with mTORC2 through its binding to RICTOR. In fact, FLNA is a substrate of mTORC2. RICTOR knockdown in GBM cells as well as PP242 treatment resulted in the inhibition of phosphorylation of FLNA. The PP242 treatment contributes to the disruption of colocalization of FLNA and actin. Taken together, these results suggest that mTORC2 affects FLNA and results in changes in motility and invasion in GBM.
Discussion
GBM has been depicted as one of the most invasive types of cancer [
2,
43]. In this paper, we have shown that PP242, an ATP competitive inhibitor of mTOR, inhibits mTORC2 and exerts dramatic effects on actin cytoskeleton and focal adhesion in GBM cells. Moreover, PP242 causes inhibition of migration, as examined by scratch wound assay, while rapamycin or MEK inhibitor has only limited effect. In addition, PP242 inhibits invasion of GBM cells. By characterizing mTORC2 purified from GBM, we have found that Filamin A (FLNA) is an mTORC2 associated protein. This is confirmed by co-IP experiments. We have also shown that FLNA is phosphorylated by mTORC2
in vitro and
in vivo. Inhibition of mTORC2 by RICTOR siRNA or PP242 treatment results in the inhibition of FLNA phosphorylation and dissociation of FLNA from actin cytoskeleton. Taken together, our results suggest that mTORC2/FLNA axis is critical for actin cytoskeleton, motility and invasion of GBM cells.
FLNA is ubiquitously expressed in non-muscle cells [
38], and this 280 kDa actin binding protein serves as an essential organizer of cell structure and function by assisting in cell shape maintenance and organization of cytoskeletal networking, providing cell plasticity, and protecting cells from shearing stresses [
44]. Because FLNA crosslinks cortical actin filaments to cell membrane, cell protrusions such as filopodia, lamellipodia, and pseudopodia can be initiated [
36,
45]. FLNA also provides connection between various cytoskeletal proteins in cytoplasm to integrin localizing on the plasma membrane. In this manner, FLNA can anchor F-actin to various transmembrane proteins [
46]. Getting released from FLNA, the integrin is activated and functional in propelling the cells [
38,
47]. Thus, the mTORC2/FLNA axis plays important roles in cellular functions relating actin cytoskeleton, motility and invasion. In addition, both FLNA and mTORC2 could affect small GTPases known to control actin cytoskeleton (Rho), and chemotaxis and accumulation of F-actin (Rac/cdc42) [
48].
By dissociating mTORC2 complex, we have shown that FLNA binds RICTOR but not mTOR. Since RICTOR is a specific component of mTORC2 (while RAPTOR is a specificity protein for mTORC1), this result explains specific effects of mTORC2 on actin cytoskeleton, motility and invasion. We further showed that RICTOR and FLNA are colocalized in GBM cells. Once associated with mTORC2, FLNA is phosphorylated by mTOR. This was shown first by demonstrating that mTORC2 purified from U87vIII cells could phosphorylate FLNA and this phosphorylation is inhibited by PP242. Second, knockdown of mTORC2 by applying RICTOR siRNA resulted in the inhibition of FLNA phosphorylation in U87vIII cells. These results suggest that FLNA is a new substrate of mTORC2. The specific site of phosphorylation, Ser2152, is located on FLNA’s repeat 20 that is involved with the migration and invasion properties of cancers [
47]. In addition, this site is associated with the binding of the protein to C-terminal region of β-integrin [
38], suggesting that multiple interactions occur at this specific location of FLNA, and pointing to the importance of Ser2152 in maintaining integrity of the protein and initiation of several cellular activities.
According to our findings, it appears that mTORC2-mediated phosphorylation of FLNA is a primary event in GBM migration and invasion, even though FLNA can be phosphorylated by multiple protein kinases including cyclic AMP (cAMP)-dependent protein kinase (PKA), p90 ribosomal kinase (RSK), PAK1, cyclin D1/Cdk4 and PKCα [
49‐
52]. Our results shown in Additional file
1: Figure S1A demonstrate that MEK inhibitor (U0126), inhibiting upstream regulators of RSK, and PAK1 inhibitor (IPA3) did not significantly affect phosphorylation of FLNA compared to PP242. While FLNA is associated with mTORC2, we did not observe PKCα association with isolated complexes (Additional file
1: Figure S1B). Taken together, it appears that mTORC2 plays critical roles in the phosphorylation event of FLNA.
We found that FLNA is overexpressed and highly phosphorylated at Ser2152 in GBM cells. This presumably reflects high level of mTORC2 activation in these cells perhaps due to overexpression of RICTOR. We have shown that phosphorylation of FLNA by mTORC2 is important for cytoskeletal effects. Inhibition of FLNA phosphorylation by ATP-competitive mTOR inhibitor (PP242) leads to impaired FLNA-actin-plasma membrane linkages and disorganization of actin cytoskeleton in addition to the disruption of FLNA-actin colocalization. FLNA appeared to move inward to the perinuclear area after being inactivated by the inhibitor. Similarly, inhibiting mTORC2 by knocking down RICTOR results in the inhibition of FLNA phosphorylation and this causes dramatic change in actin cytoskeleton and FLNA localization. From the recent study, knockdown of FLNA impairs migration of neuronal cells, disrupts actin cytoskeleton and defects filopodia formation [
53]. Therefore, both FLNA knockdown and inhibition of phosphorylation by PP242 affect actin cytoskeleton.
Our studies reinforce the idea that FLNA is a critical player in GBM. FLNA was firstly shown to be associated with P311 protein, and colocalize on the leading edges of glioma cells [
54]. Highly expressed P311 is involved with invasive glioma migration by acting downstream of β1-integrin. Disruption of β1-integrin affects P311-mediated Rac1, Cdc42 activation, and finally inhibits cell migration [
54]. Recent findings established that metastasis of GBM is related to the activity of calpain-2 enzyme, accounting for proteolysis of its substrates; Talin and FLNA through the maintenance of extracellular matrix metalloproteinases 2 (MMP2) [
55,
56]. FLNA may be significant in migration, invasion and metastasis of other cancers also. From previous studies, FLNA appears to be overexpressed in several types of cancers, such as prostate cancer, breast cancer, lung cancer, colon cancer, melanoma, and neuroblastoma [
47]. Involvement of FLNA in cancer metastasis was suggested in multiple other studies [
57‐
59]. In addition, Zhang L
et al. reported that overexpression of FLNA in
Tsc1
null neurons promoted abnormal dendritic patterning which is a common shared feature of neurodevelopmental diseases [
60].
Another protein that we found associated with purified mTORC2 is Myosin-9 (MYH9 or NMHC-IIA). MYH9 is a member of class II actin motors with ATPase activity that promote formation of lamellipodia, filopodia, and membrane ruffles [
37,
61]. This machinery is critical for various developmental processes of the cells such as embryogenesis, organogenesis, and immune synapse formation [
62]. Many kinases in several signaling pathways have been shown to regulate MYH9 [
63]. Abnormalities of MYH9 and its associated proteins cause multiple types of diseases including cancers [
62]. Also, MYH9 was reported to play important roles in breast cancer motility and glioma invasion [
64,
65]. Further work is needed to investigate whether MYH9 is a component or substrate of mTORC2 and what role it plays in mTORC2 function.
In summary, our results highlight the importance of mTORC2-FLNA lineage in glioblastoma multiforme, and suggest that FLNA is associated with mTORC2 as a new downstream target. mTORC2 activates FLNA by phosphorylating its biological regulatory site Ser2152 which elicits actin cytoskeletal changes and further affects cell motility and invasiveness. Future work should uncover signaling events involving the mTORC2-FLNA axis in GBM.
Methods
Antibodies and other reagents
Anti-FLAG M2 magnetic beads, 3X FLAG peptide, Phalloidin-FITC, anti-Vinculin (VCL), and PP242 were obtained from Sigma-Aldrich. Anti-AU1 agarose beads were from BETHYL Lab. AU1 peptide (DTYRYI) was from COVANCE. Recombinant C-terminal fragment (amino acids 1730–2639) of Human FLNA purified from E. coli was obtained from Creative Biomart. Full-length inactive recombinant AKT1/PKB was from EMD Millipore. Anti-mTOR, Anti-phospho-AKT(Ser473), Anti-AKT, Anti-phospho-FLNA(Ser2152), Anti-phospho-S6(Ser235/236), Anti-S6, Anti-phospho-ERK (Thr202/Tyr204), and Anti-ERK were obtained from Cell Signaling. Anti-FLNA was obtained from EMD Millipore. Anti-RICTOR and anti-SIN1 were obtained from BETHYL Lab. Anti-phospho-CRKII and Anti-CRKII (Ser41) were obtained from Santa Cruz Biotechnology.
Cell culture
U87 overexpressing EGFR vIII (U87vIII) and HEK293T cells were maintained in DMEM containing 10 % (vol/vol) FBS, 1 % (vol/vol) penicillin/streptomycin at 37 °C with 5 % (vol/vol) CO
2. Stable U87vIII and HEK293T cells expressing FLAG-RICTOR protein were established using lentivirus containing pRK-5-myc-RICTOR plasmid (Addgene plasmid # 1860) [
29] generated by the UCLA Vector Core Facility.
Drug treatment
U87vIII cells were cultured in DMEM containing 10 % (vol/vol) FBS, 1 % (vol/vol) penicillin/streptomycin at 37 °C with 5 % (vol/vol) CO
2 until they reached 80 % confluency. Cells were serum starved in serum-free media for 24 h prior to a 24-h treatment of different concentrations of rapamycin or PP242 in regular culturing media. We have shown that PP242 at the highest concentration (5 μM) did not inhibit ERK or PAK1 activity, confirming specific effects on mTORC2 prior to its usages in other experiments (Additional file
1: Figure S1A).
Western blotting
Cultured cells were lysed with a lysis buffer containing 0.2 % Triton X-100 and protease inhibitor cocktail (EDTA-free PIC; Roche). The amount of total protein concentration in cleared lysate was determined by Bio-Rad protein assay. Equal protein extracts from various samples were separated by electrophoresis on SDS-PAGE gel, and then transferred to a nitrocellulose membrane (GE Healthcare). The membrane was blocked in Tris-buffered saline containing 0.05 % Tween20 and 5 % bovine serum albumin, then probed with primary antibodies followed by secondary antibodies (Horseradish peroxidase (HRP)-conjugated). The blot was incubated in Pierce ECL Western Blotting Substrate solution (Thermo Scientific). Protein bands from peroxidase activities to chemiluminescent substrates were developed and detected on films.
Immunofluorescence
U87vIII cells were fixed with 4 % Paraformaldehyde, lysed with 0.2 % Triton X-100 buffer, blocked with 1 % BSA, then incubated in primary antibodies (anti-FLNA, anti-VCL, anti-RICTOR, anti-mTOR, FITC-phalloidin) overnight at 4 °C or 3 h at room temperature. Anti-mouse (Texas Red conjugate), and anti-rabbit (Alexa Flour 594 conjugate and Alexa Flour 488 conjugate) secondary antibodies from Life Technology were used. Nuclei of cells were stained by DAPI. FLNA, Actin filaments, RICTOR, and VCL, were observed using a fluorescence microscope.
Wound healing migration assay
U87vIII cells were plated onto several 35 mm collagen-coated culture dishes (Sigma- Aldrich) until the surface of each dish was covered by a monolayer of cells. A single scratch per plate was made using a small pipette tip creating a 1 mm wide linear gap. Phase contrast images of all sample groups were taken at hour 0 before drug administration. Cells were treated with PP242 2.5 μM, rapamycin 100 nM, U0126 5 μM for 12 h, then images were acquired again.
Transwell invasion assay
U87vIII cells were serum starved for 24 h prior to the experiment. One hundred thousand cells were treated with DMSO or drugs (rapamycin, PP242, or U0126) at different concentrations for 6 h in serum-free DMEM. Then, the media was changed to new 200 μl of serum-free media without drugs. Cells in drug-free media were transferred to an upper chamber of a 6.5 mm transwell insert with a 8.0 μm pore polycarbonate membrane (modified Boyden chamber) coated with 2-3 mg/ml basement matrix extract (BME) (Cultrex; Trevigen). In each lower chamber we added 500 μl of DMEM with 10 % FBS. The 24-well plates were incubated at 37 °C for 24 h. After that, DMEM and BME in the upper compartment were removed, and cells on the bottom of the membrane were washed in PBS. The membranes were then fixed in chilled methanol for 10 min following by crystal violet staining for 25 min. Cells were destained by acetic acid, and transferred to a 96-well plate. Absorbance representing relative cell numbers of each sample group was detected at 590 nm. Cell migration of each group was determined with the use of modified Boyden chamber without BME coating. Absorbance value from migrated cells of the control group (with DMSO) represented one hundred thousand cells. Absolute numbers of invaded cells under different conditions were calculated by comparing the ratio of absorbance values from invaded cells to migrated cells.
Statistical analysis
Statistical analysis of cell invasion assay was performed using absolute numbers of invaded cells from sample groups. Statistically significant differences between two data sets were assessed with the two-tailed, unpaired Student’s t-test, with P-values of P < 0.05 sufficient to reject null hypothesis.
Purification of mTORC2
U87vIII and HEK293T cells were infected with the FLAG-RICTOR lentivirus and cells stably expressing FLAG-RICTOR were obtained by puromycin selection. The drug-selected cell lines were cultured and grown, collected after washing with PBS, and stored at -80 °C for subsequent experiments. To purify mTORC2, cells were lysed in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 2 % CHAPS, 1X Complete EDTA-free protease inhibitor mixture (Roche Applied Science), and 1 mM Na3VO4). Cleared supernatant after centrifugation (16,000 × g for 10 min) was mixed with anti-FLAG M2 magnetic beads (Sigma-Aldrich) for affinity purification. The beads were collected, washed twice with wash buffer containing ATP (50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM DTT, 2 mM ATP, 0.1 % CHAPS), and washed three times with high salt wash buffer without ATP (50 mM HEPES, pH 7.4, 300 mM NaCl, 2 mM DTT, 0.1 % CHAPS). The bound proteins were eluted from magnetic beads using 3X FLAG peptide (Sigma-Aldrich) in 50 mM HEPES, pH 7.4, 500 mM NaCl, 0.4 % CHAPS. Eluted proteins were concentrated using Amicon Ultra 0.5-ml centrifugal filters NMWL 100 K (EMD Millipore, Billerica, MA). For the experiment that required dissociation of mTOR from RICTOR, 1 % Triton X-100 was substituted for 2 % CHAPS in the lysis buffer. When necessary, we included additional affinity purification to remove excess RICTOR by co-expressing AU1-mTOR and using AU1-agarose beads. This yielded mTORC2 complexes containing approximately equal amounts mTOR and RICTOR proteins. This method is called two-step purification. To perform this method, we first transfected HEK293T stably expressing FLAG-RICTOR with AU1-mTOR DNA for 48 h before cell collection. After FLAG-beads purification, mTORC2 was further subjected to another round of affinity purification using AU1 beads and eluted with AU1 peptide. Silver staining and Western blotting were performed to analyze the purified mTORC2.
Mass spectrometry
In-gel trypsin digests and LC-MS/MS
The mTORC2 preparation was fractionated by SDS-PAGE and gel slices from the 200–300 kDa MW region were excised for MS analysis. Gel slices stained with Gelcode Blue stain (Thermo Scientific) were washed with a 1:1 mixture of 100 mM ammonium bicarbonate (ABC) and acetonitrile. The gel was further destained with 100 % acetonitrile and dried down by vacuum centrifugation. Proteins in the gel slices were reduced with 10 mM DTT at 60 °C for 1 h followed by alkylation with 50 mM iodoacetamide at 45 °C for 45 min. Extensive washing with 100 mM ammonium bicarbonate and acetonitrile was performed prior to overnight trypsin digestion with 15 ng sequencing grade trypsin (Promega). The resulting tryptic peptides were extracted from the gel slices with 30 μL of 50 % acetonitrile, 1 % TFA. Extraction was repeated a total of three times and the extracted peptides were pooled prior to drying down by vacuum centrifugation. Samples were reconstituted in 15 μL 1 % formic acid in water for MS analysis.
Tryptic peptides extracted from the gel bands were analyzed by LC-MS/MS using an EASY-nLA 1000 HPLC (Thermo Scientific) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) equipped with an EASY-Spray nano-ESI source. 5 μL of each tryptic digest sample were injected onto a 75 μm X 15 cm, 3 μ, 100 Å PepMap C18 reversed-phase analytical LC column and separated with a linear gradient of 100 % solvent A (0.1 % formic acid in water) to 30 % solvent B (0.1 % formic acid in acetonitrile) over 20 min at a constant flow rate of 300 nL/min. Samples were analyzed using a Top 10 data-dependent acquisition method with 70,000 and 17,500 resolution at m/z 200 for MS1 and MS2 analysis respectively. Raw data files were processed in Proteome Discoverer (version 1.4, Thermo Scientific) using MASCOT (version 2.4.1; Matrix Science, London, UK) database searching to identify proteins entrapped in the 200–300 kDa gel slices. Tryptic peptides with up to 2 missed cleavages were searched against the SwissProt human database (2013) with the following settings: precursor and product ion mass tolerances of 10 ppm and 0.8 Da respectively, dynamic modification for oxidation (M), static modification for carbamidomethyl (C).
Co-Immunoprecipitation
U87vIII cells were lysed with 2 % CHAPS lysis buffer and centrifuged at 13200 RPM for 10 min. Cleared supernatant was divided into groups to be incubated with primary antibodies against mTOR, phospho-FLNA (Ser2152), RICTOR and RAPTOR overnight at 4 °C. Control groups have no antibody. Then, Protein G superparamagnetic beads (Dynabeads; Life Technologies) were added to each sample group and were rotated for 30-45 min at 4 °C. Immunoprecipitated and co-immunoprecipitated proteins were detected using Western blotting analysis.
siRNA treatment
On-TARGET plus Smartpool Human RICTOR siRNA was purchased from Thermo (Cat# 016984-00). U87vIII cells were transfected using Lipofectamine RNAi max (Life Technologies) and RICTOR siRNA or siGENOME-non-targeting #1 (D-001206-13-05) for 24 h before changing the media. All samples were collected 48 h after siRNA treatment. Western blotting was performed to show effects of RICTOR knockdown on FLNA phosphorylation. For immunofluorescence experiment, siRNA transfected cells were transferred to 4-well chamber slides 24 h after siRNA treatment and incubated for another 24 h. Cells were then fixed, treated with anti-FLNA, anti-RICTOR, phalloidin, followed by secondary antibodies, and were observed under the fluorescent microscope.
In vitro kinase assay
Kinase activities of mTORC2 from U87vIII cells expressing FLAG-RICTOR were examined in various conditions using affinity purified mTORC2 as described above. Purified FLNA protein (C-terminal fragment amino acid 1730–2639; Creative Biomart) from E. coli and purified AKT protein (full-length; EMD Millipore) were used as substrates for the kinase assay. In each reaction, purified mTORC2 and its substrate (FLNA or AKT) were incubated in a buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.2 mM ATP for 25 min. Negative control reactions contain either no ATP or no mTORC2. MnCl2 was added to the positive control reaction. PP242 at two concentrations, 1.25 μM and 5 μM, and 40 μM of IPA3 were added to the reactions to observe mTOR kinase inhibition. Phosphorylation of FLNA (Ser2152) and AKT (Ser473) were detected by Western blot analysis.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
N.C. and F.T. participated in the study design, data analysis and drafting of the manuscript. N.C. and P.W. performed Mass spectrometry analysis and N.C., P.W. and J.A.L. carried out data analysis. P.S.M. and J.A.L. provided suggestions on study design and discussed the data. All authors read and approved the final manuscript.