Invasiveness is one of the main hallmarks of glioblastoma (GBM) and other glial tumors, in which malignant cells diffusely infiltrate normal brain tissue and migrate along blood vessels and defined structures of the brain [1
]. This has important clinical consequences as it prevents complete surgical tumor resection, contributes to therapeutic resistance and helps to drive rapid tumor growth. Invasion is facilitated by multiple cellular processes including extracellular matrix remodeling [3
], interactions of malignant cells with the normal cells of the brain and the host immune system [4
], changes in cell adhesion properties [5
] and cytoskeletal dynamics [6
GBM is the most common malignant brain tumor and has a dismal prognosis [8
]. At present there are no clinical approaches to block GBM invasion. We previously showed that lithium chloride (LiCl) potently and specifically blocks GBM cell migration in vitro [8
]. LiCl has multiple targets within the cell [9
], including the protein kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is ubiquitously expressed in mammalian cells, with two closely related isoforms, GSK-3α and GSK-3β which have distinct and overlapping functions [12
]. GSK-3 is a multi-tasking enzyme, known for its role in the regulation of glycogen synthesis, via inactivation by Akt, and also for its role as a critical mediator of Wnt signaling [15
]. GSK-3 has been associated with a number of processes involved in cell motility via interactions with cytoskeletal components [11
In pre-clinical murine GBM xenograft models we showed that protein kinase inhibitors of the indirubin family, which are known to potently inhibit GSK-3, block invasion and angiogenesis improving survival [17
]. However, we have not yet defined the mechanisms underlying our observations in GBM cells. Here, we performed a proteomic study, which revealed consistent and highly significant downregulation of the intermediate protein vimentin after treatment of GBM cells with LiCl, and the indirubin derivative 6-bromo-indirubin-3-oxime (BIO).
Vimentin is a major component of the intermediate filament cytoskeleton and is best known in cancer as a marker of cellular epithelial to mesenchymal transition (EMT) [9
], a phenomenon associated with cancer cell invasion and metastatic tumor spread. Vimentin is known to play an important role in maintenance of cellular integrity and resistance to stress, and vimentin knockout mice show impaired wound healing ability [19
]. Vimentin has been shown to regulate cell migration via interaction with paxillin, integrin α6β4 and myosin II [20
]. The role of vimentin in GBM has not been explored in detail regardless of the fact that it has been used as biomarker [21
Here we show that LiCl and BIO, which block GBM cell migration, both affect the dynamic regulation of vimentin in GBM cells, and that vimentin knockdown partially blocks GBM migration. We also show that vimentin and GSK-3 physically associate. Thus, vimentin may affect GBM invasion downstream of anti-invasive compounds LiCl and indirubin derivatives.
Our previous studies implicated the conserved, ubiquitous and multi-tasking protein kinase GSK-3 in GBM cell migration [8
]. Here we performed a proteomics screen to investigate the mechanisms underlying these observations, and show for the first time that GSK-3 plays a role in regulation of the intermediate cytoskeleton component vimentin. Specifically, we establish that (1) GSK-3 regulates vimentin dynamics, (2) GSK-3 interacts with and phosphorylates vimentin, (3) vimentin facilitates GBM cell migration, and (4) vimentin is highly expressed in GBM patient specimens, and is prognostic, particularly in lower grade gliomas.
This study was performed as a development from our previously published observations on small molecules with potent inhibitory effects on GBM cell migration [8
]. First, we showed that LiCl was a potent and reversible inhibitor of GBM cell migration in vitro [8
]. These effects were observed at 10–20 mM LiCl, which exceeds the maximum tolerated dose of around 1 mM. LiCl is known to inhibit GSK-3 [25
], therefore we went on to show that GSK-3α/β siRNA could block GBM migration and that other small molecule GSK-3 inhibitors could block GBM cell migration. During these studies, we examined derivatives of the Chinese medicine indirubin, which are known to inhibit GSK-3 [26
]. These molecules blocked migration and angiogenesis in vivo in murine GBM models, improving animal survival [17
To further study the underlying mechanisms, here we examined the impact of LiCl on GBM cells using proteomics-based approach. 2D-gel electrophoresis/mass spectrometry identified several proteins significantly altered by LiCl treatment. Interestingly these were all involved with cytoskeleton regulation and merit further study in the context of GBM cell migration. Indeed, one of these proteins, CRMP-4, regulates microtubule dynamics in axon outgrowth, and is also a known GSK-3 substrate [14
]. Vimentin was the most significantly altered protein and the focus of this study. Additionally, by analysis of patient specimens by Western blotting and immunofluorescence, as well gene expression databases and the human protein atlas, we showed that vimentin is highly abundant in most GBM cases. At the mRNA level it is significantly prognostic in GBM and even more so in astrocytoma where the distribution of vimentin expression appears much broader than in GBM. Further studies are needed to determine whether the reduced expression in astrocytoma compared with GBM is at the tumor cell level, or rather reflects lower cellularity in lower grade tumors. To determine whether vimentin is truly a useful prognostic marker, much more detailed studies are required with larger well-characterized patient populations.
Our data suggest that a primary target of the anti-invasive drugs is vimentin dynamics. This is supported by the observations that (1) detergent soluble vimentin levels are rapidly reduced upon exposure to either LiCl or BIO, and (2) FRAP studies which showed a reduction in the ability of the vimentin cytoskeleton to recover after photobleaching of a GFP-vimentin fusion protein. Presumably this prevents the cell undergoing the restructuring and dynamic alterations required for effective migration and invasion.
Structurally, vimentin is a 57 kDa protein with a highly conserved α−helical rod domain flanked by N- and C-terminal domains which associate to ultimately form intermediate filament networks. Phosphorylation of vimentin, largely at the N-terminus, regulates its ability to form filaments and several kinases have been implicated in this process including protein kinase A (PKA), aurora kinase B and Akt which phosphorylate vimentin at various N-terminal sites [11
]. Our data strongly implicates GSK-3 as a candidate vimentin kinase, although we have not yet definitively shown this. Analysis of the vimentin amino acid sequence reveals the presence of GSK-3 phosphorylation sites (Ser 50) [14
] at the N-terminus, which are under investigation.
Further studies on the role of vimentin in GBM are warranted. This protein is highly abundant in GBM and is involved in multiple cellular processes including cell migration. However, one challenge we faced throughout this study was that vimentin manipulation in GBM cells is difficult; sustained knockdown was not straightforward due to the high levels of vimentin in GBM cells, and we have so far been unable to generate CRISPR gene edited GBM cell lines null for vimentin. Additionally, further detailed molecular studies are needed to understand better the effects of vimentin on GBM cell migration. Our data also shows that proteomics can be a very effective approach to identifying molecular changes relevant in GBM cell migration, revealing new targets and mechanisms.
4. Materials and Methods
4.1. Cell Lines, Tissue Samples and Chemicals
The U87 cell line was provided by Dr Webster Cavenee (Ludwig Institute for Cancer Research, La Jolla, CA, USA). U251 and U373 cells were purchased from ATCC (Manassas, VA, USA). All cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% FBS and 1% penicillin-streptomycin. The GBM stem-like cells (GSCs) were derived from fresh samples of surgically resected brain tumors obtained from The Ohio State University Tissue Procurement Shared Resources under a protocol approved by OSU IRB. GSCs were maintained as neurosphere suspension cultures in Neurobasal medium with 20 ng/mL fibroblast growth factor (FGF) and epidermal growth factor (EGF). Unless otherwise stated, reagents, chemicals and supplies were purchased from Thermo-Fisher Scientific (Waltham, MA, USA).
4.2. Western Blotting
Proteins from 5 × 106 cells were isolated by 15 min extraction in TX100 lysis buffer: 1% Triton X100, 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM β-glycerophosphate supplemented with 50 mM NaF, 1 mM PMSF, 1 mM DTT, 2 mM Na3VO4, 10 μg/mL Leupeptin, 10 µg/mL Aprotinin, 10 μg/mL Pepstatin, 0.25 ng/mL Microcystin LR (EMD Millipore, Billerica, MA, USA). To achieve total solubilization of vimentin fibers, TX100 lysis buffer was supplemented with 8 M urea. Tissue samples were solubilized by sonication in urea containing TX100 lysis buffer. For Western blotting, proteins (100 μg) were separated by SDS-PAGE (Criterion system, Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose (Bio-Rad). Antibodies used for Western blotting were: mouse anti-vimentin V9 (MS-129-P, Thermo Scientific, (Waltham, MA, USA) mouse anti-GSK-3αβ (368662, EMD Millipore), mouse anti-α-tubulin (T6074, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-GAPDH (ab9484-200, Abcam, Cambridge, MA, USA) and peroxidase-conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA).
4.3. Cell Migration Assays
For vimentin knockdown, 200 pmols of Smartpool siRNA, or scrambled siRNA control (Dharmacon, Chicago, IL, USA) was introduced into cells with Lipofectamine 2000 (Thermo Scientific) in a six-well plate 24 h prior to the transwell assay (modified Boyden chamber assay [28
]). Assays were performed using 8 μm pore size inserts (ISC Bioexpress, Kaysville, UT, USA), and 50,000 cells per well. Cells were allowed to migrate for 6 h prior to fixation in 1% glutaraldehyde and cell visualization was performed by staining with 10 µg/mL 4′,6-diamidino-2-phenylindole (DAPI). Images of the whole membrane were captured with a motorized Nikon Eclipse Ti microscope system. The time-lapse version of the transwell assay was performed with FluoroBlock inserts (Corning cat# 351152 & 353504) and U251pCDH (copGFP labeled cells, System Biosciences plasmid pCDH-EF1-MCS-T2A-copGFP). Images of the whole insert were captured with a motorized Nikon Eclipse TE2000 microscope system (Nikon, Tokyo, Japan) equipped with an on-stage incubator with CO2
and temperature control. The invasion/migration assay performed in collagen gel was performed as described previously [8
]. Analysis and quantification of signal was performed in ImageJ (https://imagej.nih.gov/ij
], Microsoft Excel was used for data handling and Prism Graphpad version 8 (San Diego, CA, USA) was used for data presentation.
4.4. Two-Dimensional Fluorescence Difference Gel Electrophoresis
Whole cell proteomic analysis was performed by using 2-D fluorescence difference gel electrophoresis (DIGE) recommended by The Ohio State University Proteomics Shared Resource https://www.ccic.osu.edu/msp-proteomics
]. U251 cells (1 × 107
) were incubated with 20 mM NaCl or 20 mM LiCl for 24 h. Cell pellets were snap frozen and processed for DIGE analysis by The Ohio State University Mass Spectrometry and Proteomics Facility using the Ettan DIGE system. After 2D-SDS-PAGE, gels were imaged with the Typhoon Variable Mode Imager. Image analysis and quantitation were performed using DeCyder Differential Analysis Software (GE Healthcare, Atlanta, GA, USA). Selected spots were excised and processed for protein/peptide identification.
4.5. Fluorescence Recovery After Photobleaching (FRAP) Assay
U251 cells were stably transfected with a pEGFP-vimentin expression vector, kindly provided by Professor Robert Goldman, Northwestern University [31
]. High magnification, time-lapse image series of GFP-vimentin-labeled cells were collected using a Zeiss LSM510 confocal microscope system (Carl Zeiss Inc., Thornwood, NY, USA), equipped with an in vitro cell culture on-stage incubator (PeCon GmbH, Erbach, Germany). Images were collected every 10 s, for 20 min with minimal laser power (1%). A 5 μm2
area was burned with 100% laser power for 2 s (until the fluorescence from the selected area was equal to background levels). The half-time of fluorescence recovery T1/2 was analyzed using Zeiss LSM510 control software v3.2 (Jena, Germany). For image dynamic measurement 200-seconds-long, 50 frames sequences were collected. A frame-to-frame difference was calculated with ImageJ/Image Calculator function; data is presented as percent value of first frame of time sequence, accounting for background/noise. Co-localization of GSK-3 with vimentin was assayed on the same microscope system; U251 cells were transiently transfected with pcDNA4TO/CFP-hVimentin (NM_003380) and pcDNA4TO/YFP-GSK-3α/β plasmids. The pcDNA4TO plasmid was purchased from Thermo-Fisher/Life Technologies (cat# V1020), human vimentin was obtained from OriGene (SC111054), GSK-3α/β coding sequences were provided by Dr. Chris Phiel, Nationwide Children’s Hospital, Columbus, OH, USA.
4.6. GST Pull-Down Assay
U251 cells were transiently transfected with pDEST27/GST (control) and pDEST27/GST-GSK-3 (either α or β isoform) expression vector. A total of 500 μg of TX100 protein extract was incubated with 50 μL of glutathione agarose beads (Invitrogen, Carlsbad, CA, USA) at 4 °C for 1 h. Beads were extensively washed with TX100 lysis buffer and pulled-down proteins were extracted by boiling in 2× Laemmli Western blot sample buffer (Bio-Rad) and analyzed by Western blotting.
4.7. Digital Data Resources