Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein
MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6 % of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named METΔ7−8. METΔ7−8 is located predominantly in the cytosol and is constitutively active. The auto-activating nature of METΔ7−8, in combination with a lack of transmembrane localization, renders METΔ7−8 not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.
KeywordsMET Glioma Mutation Protein localization Genetic deletion Auto-active Intracellular location Biomarker
The MET proto-oncogene (chromosome 7q31.2) encodes the tyrosine kinase membrane receptor MET (also called Scatter Factor Receptor), which is essential during development. Signaling from the receptor controls epithelial-to-mesenchymal transition (EMT) of myogenic precursor cells during differentiation into skeletal muscle cells , a process that involves migration over long distances in the embryo. In adults, MET is involved in tissue regeneration upon injury .
MET is produced as a glycosylated single-chain precursor protein of ~190 kDa which, during transport to the membrane, undergoes furin-mediated cleavage in the trans-Golgi network [8, 29]. The resulting mature receptor consists of an extracellular 50 kDa α-chain, covalently attached via a disulfide bond to a membrane-spanning 140 kDa β-chain [20, 56]. The extracellular segment consists of an N-terminal Sema domain that is involved in ligand binding, a small cysteine-rich domain, and four IPT (Immunoglobulin-like fold shared by plexins and transcription factors) domains, which connect the Sema and cysteine-rich domains with the C-terminal β-subunit .
Upon binding of the ligand hepatocyte growth factor (HGF, scatter factor), receptor dimerization occurs followed by trans-phosphorylation in the intracellular tyrosine kinase (TK) domain at tyrosine (Y) residues 1230, 1234 and 1235 [14, 34]. The TK domain subsequently induces auto-phosphorylation of Y1349 and Y1356, which act as docking sites for signal transduction molecules including GAB1, GRB2, phospholipase-C and SRC . Phosphorylated GAB1 interacts with molecules like PI3-K and SHP2, which together induce several downstream signaling pathways. MET signaling is mediated by, among others, the PI3-K/AKT and RAS/MAPK pathways, which induce cell cycle progression, survival, cytoskeletal changes and invasion (reviewed in ). In addition to its role downstream of HGF, MET can also be involved in signaling of other transmembrane receptors, including VEGFR2, CD44v6, EGFR and Plexin B1 [19, 26, 35, 44]. Upon ligand-induced receptor dimerization, MET is internalized via endocytosis and may be recycled . Phosphorylation of Y1003 in the juxtamembrane (JM) domain of the receptor leads to ubiquitination and subsequent proteasomal degradation . Thus, levels of MET in the cell are tightly regulated.
Aberrant activation of MET signaling is a tumor-promoting event in a variety of malignancies and can be induced by several mechanisms, including alternative mRNA splicing, exon skipping and crosstalk with other receptors . In high-grade gliomas, the frequently occurring oncogenic EGFR mutant EGFRvIII can induce overexpression of both MET and HGF, a process that is balanced by wild-type EGFR activation . MET amplifications have been found in a number of tumor types including glioblastoma (GBM) [9, 10] and missense mutations in the Sema, the TK and the JM domain have been reported to affect HGF binding, kinase activation and receptor degradation, respectively [1, 30, 32, 36, 38, 43, 48, 49]. Recently, gene fusions between the protein tyrosine phosphatase PTPRZ1 and MET, resulting in constitutive activation of MET, were described in 16 % of secondary GBMs . Activation of MET signaling has been proposed as a mechanism of resistance to EGFR inhibitors, likely a result of the similarities in downstream signaling events from both receptors .
The significant role that MET plays in tumor progression and metastasis has made it a prime therapeutic target in oncology. MET tyrosine kinase inhibitors and therapeutic antibodies against the extracellular domain of MET and against HGF, all preventing HGF-mediated MET activation, are currently in clinical trial (www.clinicaltrials.gov). In a previous study, we have shown that the combined VEGFR2/MET tyrosine kinase inhibitor cabozantinib (XL-184, CoMETRIQ) potently inhibits MET phosphorylation, cell proliferation and migration and consequently prolongs survival of mice carrying orthotopic E98 glioma xenografts . Here, we identify a novel intragenic MET deletion in E98 cells, which results in a truncated protein that is constitutively active and lacks membranous expression, thereby having important implications for therapeutic strategies targeting MET. We show that this mutation occurs in 6 % of glioblastomas and, like the EGFR mutation EGFRvIII , is relatively specific for this tumor type.
Materials and methods
Immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue sections was performed as previously described using antibodies against MET and P-MET (clone D1C2 and D26, respectively, both CST) . Antibodies were visualized via sequential incubations with biotinylated secondary antibodies, avidin–biotin complexes (Vector laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine solution (Power-DAB, ImmunoLogic, Duiven, The Netherlands).
The E98 cell line and xenograft model and genetic analysis thereof have been described before [12, 42]. E98, U87, A549, HEK-293T and TOV-112D or TOV-112D-MET cells  were cultured in DMEM + 4.5 g/l glucose medium (PAA Laboratories, Pasching, Austria) supplemented with 10 % fetal calf serum (FCS) (PAA) and gentamycin (40 μg/ml). All cell lines were maintained at 37 °C in the presence of 5 % CO2. To examine HGF-induced MET activation, E98 and A549 cells were seeded in 6 wells plates. The next day, cells were serum-starved overnight, followed by a 10 min treatment with 50 ng/ml HGF (Miltenyi Biotec, Bergisch Gladbach, Germany). In some experiments, prior to HGF incubation cells were incubated with the anti-MET llama VHH G2  or cabozantinib (XL-184, Exelixis, San Francisco, CA, USA) for 60 min.
Genetic analysis of E98
Genomic DNA from E98 cells was analyzed by semi-conductor sequencing (IonPGM, Life Technologies) using the comprehensive cancer panel (Life Technologies) that targets 409 cancer-related genes. The IonPGM E98 library generation was performed according to the manufacturer’s protocol. In short, 10 ng of DNA per pool was amplified in 21 cycles by PCR using the Ion AmpliSeqTM mastermix, followed by barcode and adapter ligation. Amplified products were purified with Agencourt AMPure XP beads (Beckman Coulter Genomics, High Wycombe, UK). The library was diluted to 20 pM. Emulsion PCR was performed using the Ion OneTouchTM 200 Template kit following the protocol of the Ion OneTouchTM System. Next, Ion Sphere Particles (ISPs) were recovered and enriched for template-positive ISPs using Dynabeads MyOne Streptavidin C1 beads (Life Technologies) in the Ion OneTouchTM ES instrument (Life Technologies). ISP enrichment was quantified using the Qubit 2.0 fluorometer (Life Technologies). Sequencing primer and polymerase were added to the final enriched spheres before loading onto an Ion 318 chip according to the Ion PGMTM 200 sequencing kit protocol. The gene copy number analysis was performed as follows. The relative number of sequence reads aligned to a specific gene was determined by dividing by the total number of aligned reads of E98, and was divided over the relative number of sequence reads of the same gene in non-neoplastic blood cells. The relative ratios are plotted in a graph based on the genomic position of the gene.
To perform confirmative mapping of the intronic deletion in MET on the nucleotide level, genomic DNA was isolated from E98 and U87 cells using the DNeasy Blood & Tissue Kit (QIAGEN), according to the manufacturer’s protocol. 10 ng was subjected to PCR, using primer MET1997Fw (5′-CTCCTTGGAAATGAGAGCTG-3′, forward, exon 6, genome location chr7q;g.116395517-36) and reverse primer MET2414Rev (5′-GGGATCTTCACGGTAACTG-3′, located in exon 9, CHR7q; g.116398565-45). Human genome sequence annotations are in all cases based on assembly hg19.
Formalin-fixed, paraffin-embedded sections (4 μm) on SuperFrost glass (dried >45 min at 56 °C) were deparaffinized and rehydrated in ddH2O. After boiling in a microwave in sodium citrate buffer (pH 6), slides were allowed to cool to RT, washed in ddH2O and incubated for 5 min in 10 mM HCl. Proteins were digested with pepsin (200 U/ml, Sigma) for 15 min at 37 °C. Subsequently, slides were rinsed in 10 mM HCl and PBS and postfixed for 5 min in 1 % paraformaldehyde (PFA, Merck)/PBS. Sections were washed in ddH20, dried and hybridized with 10 μl probe mix (1 μl cep7 Spectrum Green (06J37007) + 1 μl LSI MET Spectrum Red (06N05-001) + 7 μl hybridization buffer in MQ, all Vysis) under a cover slip. Sections were denatured at 80 °C for 10 min, followed by hybridization o/n at 37 °C in a Hybridizer (Dako). After removing the coverslip by soaking for 5 min in 2× SSC buffer (Maxim Biotech) at 42 °C, slides were washed 3 times in 2x SSC buffer at 73 °C, once in 2xSSC (5 min) and once in ddH2O. After dehydration in EtOH, slides were air-dried in the dark and mounted in Vectashield/DAPI (3 parts Vectashield/DAPI + 1 part Vectashield, all vector). Slides were analyzed on a Leica Fluorescence microscope.
RT-PCR and cloning
RNA was isolated from cell lines E98 and TOV112D-MET  using TRIzol Reagent (Life Technologies) and reverse transcribed with MMLV-RT (New England Biolabs) using oligo-dT primers, according to the manufacturer’s instruction. HGF was PCR amplified from cDNA using Phusion High-Fidelity DNA polymerase (Finnzymes, ThermoFisher Scientific, Waltham, USA) and primers HGF-Fw204 (5′-CTGCAGCATGTCCTCC TGCA-3′) and HGF-Rv504 (5′-GAGGTCAAATTCATGGCCAA-3′) (30 cycles, annealing at 55 °C, 20 s; extension at 72 °C, 20 s). Control PCR reactions were performed for housekeeping gene HMBS (Hydroxymethylbilane Synthase) using primers p361-Fw (5′-TGCCAGAGAAGAGTGTGGTG-3′) and p425-Rv (5′-GTTAAGCTGCCGTGCAACATC-3′).
MET open reading frames were PCR amplified from cDNA using Phusion DNA polymerase and primers flanking start and stop codons (MET173EcoR1-Fw: 5′-CGAATTCGATAAACCTCTCATAATGAAGG-3′ and MET4421NotI-Rv 5′-AGCGGCCGCCTATGATGTCTCCCAGAAGG-3′). PCR products were purified on agarose gel, digested with NotI and cloned as blunt-NotI fragment in pIRESneo-EcoRV-NotI (Clontech Laboratories, Inc, CA, USA) to yield pIRESneo-MET and pIRESneo-METΔ7–8. Full sequences were obtained by Sanger sequencing via the sequencing facility of Radboud UMC.
The extracellular parts of MET were PCR amplified from E98 and TOV-112D-MET cDNA using primers MET173EcoR1-Fw and MET3028NheI-Rv (5′-CGCTAGCCTGATCTGGTTGAACTATT AC-3′), and cloned in vector pHLsec-BAPHIS, a derivative from pHLsec-HIS (Addgene, Cambridge, MA, USA). The resulting vector adds a biotin acceptor peptide and His-tag to the carboxyterminus of the MET extracellular domains, resulting in pHLsec-METED-BAPHIS and the E98-derived variant pHLsec-METΔ7–8ED-BAPHIS.
Transfection and protein purification
pIRESneo-MET or pIRESneo-METΔ7–8 were transfected into HEK-293T or TOV-112D cells in 6-well culture dishes (Greiner Bio-One, Krëmsmunster, Austria) using Fugene HD transfection reagent (Promega, Fitchburg, WI, USA) according to the manufacturers’ instructions. After 48 h, cell monolayers were washed with PBS and cell extracts prepared in RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling Technology, CST, Danvers, MA, USA).
In separate experiments, pHLsec-METΔ7–8ED-BAPHIS or pHLsec-METED-BAPHIS were cotransfected in a 1:1 ratio with the biotin-ligase expression construct pDISPLAY-BirA-ER (Addgene) in HEK293T cells and cells were cultured in the presence of 10 μM biotin (Sigma-Aldrich, St Louis, MO, USA). After 48 h, cytosolic extracts were made in RIPA buffer, and medium (1 ml) was mixed with 100 μl Ni–NTA Sepharose slurry (IBA, Goettingen, Germany). After a 1 h incubation at 4 °C, Ni-beads were washed with buffer (500 mM NaCl, 50 mM phosphate buffer, pH 7.4) and loaded onto a poly-prep column (Bio-Rad, Hercules, CA, USA). After washing off specifically bound proteins with 2 ml 10 mM imidazole, His-tagged an biotinylated MET extracellular domains were eluted with 0.5 ml 0.5 M imidazole, and dialysed o/n at 4 °C to 50 mM TRIS/150 mM NaCl pH 7.5.
MET downregulation via short hairpin RNAs
To create MET knockdown constructs, shMET1 (GTATGTCCATGCCTTTGAA) and shMET2 (GTATGTCCATGCCTTTGAA) oligonucleotide heteroduplexes were ligated in pENTR/U6 vector and subsequently Gateway-cloned into pLenti6/BLOCK-iT-DEST-TagRFP according to the manufacturer’s protocols (Invitrogen). Generation of lentiviruses was done in HEK293FT cells as described previously .
RIPA extracts (20–40 µg protein per lane) and purified His-tagged proteins from conditioned media (equivalent of 20 µl conditioned medium) were subjected to PAGE on 8–10 % polyacrylamide gels and western blotting according to standard procedures. Nitrocellulose blots (Whatman Optitran BA-S85, GE Healthcare, Little Chalfont, UK) were blocked with PBS/Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) (1:1), followed by overnight incubation with primary antibodies at 4 °C. Antibodies used were against MET N-terminus (clone EP1454Y, Epitomics, Abcam, Cambridge, UK), MET C-terminus (clone D1C2), phosphorylated (P)-MET (Y1234/1235, clone D26), P-AKT (S473, clone D9E), P-ERK1/2 (T202/Y204, clone 20G11) (all CST), GAPDH (clone 6C5, Abcam), and α-tubulin (clone 236-10501, Molecular Probes, Life Technologies). Biotin groups and primary antibodies were visualized using, respectively, streptavidin-680 (Molecular Probes, Life Technology) and appropriate secondary antibodies [goat-anti-rabbit-IRDye800 (Rockland Immunochemicals, Gilbertsville, PA, USA) or Alexa Fluor 680 goat-anti-mouse IgG (Molecular Probes, Life Technologies)]. Blots were scanned on the Odyssey imager (LI-COR Biosciences).
Protein domain modeling
A homology model for the new hybrid IPT-domain using the WHAT IF & YASARA Twinset was generated [31, 60]. We used the experimentally solved 3D structure 2uzx, which contains the human tyrosine kinase MET. The first 60 residues of the hybrid domain are identical to this structure, whereas the following 38 residues were modeled based on homology between the 2 domains.
E98 and U87 cells, grown on Nunc Lab-Tek chamber slides (Sigma-Aldrich) to 40 % confluence, were fixed with 2 % PFA in 0.1 M phosphate buffer (pH 7.4) for 15 min at RT, followed by three washes with PBS, 30 min glycine incubation (100 mM in PBS) to quench PFA-induced autofluorescence, three PBS washes and permeabilization with digitonin for 10 min (100 μM in PBS). Aspecific binding was blocked by incubation with 20 % normal goat serum in PBS/1 %BSA for 20 min, and cells were incubated overnight at 4 °C in a humidified chamber with PBS/1 %BSA containing (combinations of): mouse monoclonal anti-CD44 antibody (as a surface marker, clone Hermes-1, ThermoFisher Scientific); rabbit anti-MET (clone D1C2, CST); rabbit anti-P-MET (clone D26, CST); mouse anti-EEA-1 (clone 14/EEA1, BD Biosciences, early endosome marker); anti-CLIMP-63 (clone G1/296, rough ER marker, a kind gift of J. Fransen). Primary antibodies were detected using Alexa Fluor 488- or 594-labeled secondary goat-anti-mouse and Alexa Fluor 488- or 568-labeled goat-anti-rabbit IgGs (all Life Technologies), 1:200 diluted in PBS/1 %BSA. Q-nuclear deep red (1:200, Life Technologies) was used to stain nuclei, and cells were mounted in Fluoromount G with DAPI (Southern Biotech, Birmingham, AL, USA). Cells were analyzed using a confocal laser scanning microscope (Leica SP2 CLSM) and Leica confocal software.
E98 and A549 cells were allowed to adhere and grown to 80 % confluence in a 6-well plate (Cellstar, Greiner Bio-One, Kremsmünster, Austria). Cells were washed 3 times with ice-cold PBS and incubated for 30 min at 4 °C with 0.5 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (ThermoFisher Scientific). This compound does not penetrate cells and biotinylation is therefore confined to exposed membrane proteins. In parallel, Tris-free RIPA extracts, prepared from equivalent numbers of E98 or A549 cells by repeated washing over Amicon Ultra K10 centrifugal filters (Merck Millipore, Billerica, MA, USA), were treated similarly to biotinylate all cellular proteins. The reaction was quenched by washing cells or cytosolic proteins three times with 100 mM glycine/PBS and two times with PBS (using K10 columns in the case of protein lysates). Intact biotinylated cells were then subjected to lysis with RIPA buffer. Protein concentrations in all lysates were determined using the BCA protein concentration assay (ThermoFisher Scientific). MET was immunoprecipitated from 200 µg total protein in a volume of 200 μl using anti-MET (1:50, clone D1C2, CST) for 1 h at 4 °C. Immune complexes were captured by incubation for 30 min at 4 °C with 10 μl prot A agarose slurry (Roche Diagnostics, Basel, Switzerland), followed by centrifugation (14,000 rpm, 4 min) and three PBS washes. Immune complexes were solubilized by heating (5 min 95 °C) in 30 μl 2× SDS-PAGE sample buffer (0.2 % SDS, 62.5 mM Tris–HCl pH 6.8, 10 % glycerol and 0.2 μM DTT). Samples were subjected to 10 % SDS-PAGE and western blotting as described above.
Glioma tissues (n = 80) were obtained from the Radboud UMC and from the University of Cologne. Sarcoma (n = 25) and castration-resistant prostate cancer (CRPC) tissues (n = 43) were obtained from Radboud UMC. RNA was isolated from tissues using the mirVANA RNA isolation kit (Life Technologies) according to standard procedures. For cDNA synthesis, 1 μg RNA was reverse transcribed using the Quantitect Reverse Transcription Kit (QIAGEN, Venlo, The Netherlands) using hexanucleotides. To distinguish wtMET from METΔ7−8-transcripts, a PCR was performed in AmpliTaq Gold 360 mastermix (Applied Biosystems, Life Technologies) using primer MET1997Fw (5′-CTCCTTGGAAATGAGAGCTG-3′, forward, located in exon 6), and primer MET2342Rv (5′-CAGTTGAAATGGTTTGGGCTG-3′, reverse, located in exon 9). This PCR results in a 105-bp product for METΔ7–8 and a 345-bp product for wtMET. Conditions were: denaturation 95 °C, followed by 35 cycles of 95 °C denaturation; annealing at 58 °C, 30 s; elongation at 72 °C, 1 min. A final elongation step of 7 min was done at 72 °C.
E98 MET protein is auto-activated in an HGF-independent fashion
E98 cells express a truncated version of an amplified MET gene
METΔ7–8 is aberrantly processed
MET is cleaved by the endoprotease furin, which is localized predominantly in the trans-Golgi network, but also in vesicles and near the plasma membrane [8, 29, 52]. To test whether the inefficient cleavage of METΔ7–8 in E98 cells is related to intracellular transport defects, we analyzed the subcellular localization of METΔ7–8 in detail via confocal microscopy. METΔ7–8 did not co-localize with the cell surface marker CD44 and was confined to the cytosol (Fig. 4b). In contrast, in U87 cells METwt did co-localize with CD44. Additional intracellular staining in U87 cells reflects de novo synthesized material that is being processed for constitutive secretion. Immunostainings with the early endosome marker EEA-1 and the rough endoplasmic reticulum (RER) marker CLIMP-63 suggested that METΔ7–8 in E98 cells is predominantly retained in the RER (Fig. 4c).
To confirm the absence of cell surface expression of METΔ7–8 on E98 cells, we labeled intact E98 cells or cell lysates with NHS-biotin and immunoprecipitated MET using specific antibodies, followed by SDS-PAGE/Western blot and staining for biotin and MET. Whereas METs’ N-terminal α-chain was readily biotinylated in E98 cell lysates, no detectable MET biotinylation occurred when intact cells were labeled (Fig. 4d, lane 1). In contrast, biotinylated MET was readily detected in A549 cells upon labeling of intact cells, as shown by the biotin-labeled α-chain (Fig. 4d, lane 3). Thus, these data confirm that METΔ7–8 is predominantly localized intracellular and is poorly exposed on the cell surface of E98 cells.
To further confirm a defect in intracellular trafficking of METΔ7–8, we analyzed secretion patterns of extracellular domains of wtMET or METΔ7–8 (ending with residue D929, numbering according to MET variant 2 (NP_000236.2), containing a C-terminal biotin tag. Whereas the extracellular domain of wtMET was properly processed and secreted into the culture medium (as illustrated by the presence of the 309-929 biotinylated extracellular β-chain and the MET25-308 α-chain, Fig. 4e, lane 4), no secreted MET products were found in medium of cells, transfected with the METΔ7–8 ectodomain (Fig. 4e, lane 3). Instead, all biotinylated MET products were located intracellularly (Fig. 4e, lane 1).
185-kDa METΔ7–8 is not affected by antagonistic anti-MET antibodies but is inhibited by cabozantinib
Prevalence of METΔ7–8
Data mining of the COSMIC and TCGA databases did not uncover the intronic deletion in gliomas and other tumor types (not shown). Intragene deletions spanning multiple exons are, however, in general more difficult to recognize in whole-exome sequencing (WES). Furthermore, the coverage of the exact location of the deletion is poor in the mostly used WES protocols, and as can be seen in Fig. 2b, even targeted sequencing reveals the mutation only after detailed bioinformatic analysis. We, therefore, assume that detection of METΔ7–8 requires dedicated PCR protocols.
METΔ7–8 expression in glioma, sarcoma and CRPCs
6/102 (5.8 %)
2/5 (40 %)
2/16 (12.5 %)
2/61 (3.3 %)
Sanger sequencing of the smaller PCR products of one patient with glioma confirmed the deletion of exons 7 and 8 (not shown). Of note, due to the lack of high-quality genomic DNA from these clinical samples we could not discriminate in these samples whether METΔ7–8 resulted from a mutation or from exon skipping or alternative splicing, such as suggested for MET variants lacking exon 10 or 14 .
A major problem in glioma treatment is diffuse growth in the neuropil, and it has been suggested that the MET oncogene is causally involved in this phenotype in subsets of tumors . Several signal transduction pathways that are induced by MET activation are shared with other tyrosine kinase receptors and it is increasingly recognized that activation of MET bypasses the need for EGFR activation, generating resistance to EGFR-targeted therapies . Also, there is an interesting crosstalk between MET and EGFR: the EGFR variant III (EGFRvIII) stimulates expression of both MET and its ligand HGF, possibly contributing to oncogenicity of EGFRvIII , but this phenomenon is counteracted by EGF-mediated activation of wild-type EGFR in the same complex .
We previously reported that MET is crucially implicated in proliferation, survival and migration of (EGFR-negative) E98 glioma cells in vitro. To further investigate the involvement of MET in the biological behavior of E98 cells, we attempted to knock down the protein by a lentiviral shRNA approach. Attempts to generate stable MET knockdowns using 2 different MET shRNAs failed since both shRNA encoding lentiviruses were lethal to E98 cells, in contrast to control viruses (data not shown). In agreement with a crucial role of MET for cell survival, we showed that treatment with the MET tyrosine kinase inhibitor cabozantinib prolongs survival of mice carrying orthotopic E98 xenografts . Based on the observation that the pattern of MET phosphorylation in E98 xenografts was remarkably homogeneous in the absence of its ligand HGF, we analyzed the MET product in E98 cells in more detail and found that the protein is expressed as a truncated product, which lacks effective furin cleavage and is predominantly retained intracellular in its active, phosphorylated form. The truncation generates a novel IPT domain consisting of a fusion between the carboxyterminus of IPT1 and the amino terminus of IPT2. Modeling of the novel fusion IPT domain suggests that it adopts a similar structure as the IPT domains in wild-type MET, although a small stretch of extra amino acid residues is accommodated in a loop, extending towards the Sema domain. Since this loop approaches the furin cleavage site in the Sema domain, it may directly affect furin cleavage. Interestingly, the colorectal adenocarcinoma cell line Lovo lacks furin protease and consequently cannot properly process MET . In this cell line, the unprocessed preform is still expressed on the cell surface, binds HGF and is capable of signaling. Thus, defective processing alone does not explain intracellular retention of METΔ7–8 and we therefore hypothesize that the novel IPT1-2 fusion domain in METΔ7–8 is involved in defective furin cleavage, intracellular retention of the uncleaved perform and auto-activation of the uncleaved preform.
Recently, effects of various activating mutations in the MET TK domain have been analyzed in detail. These mutants are expressed on the cell surface but are subject to increased rates of turnover, resulting in accumulation in early endosomes where they can still signal . METΔ7–8 did not accumulate in early endosomes, suggesting that the underlying mechanism of intracellular retention is different . Our confocal microscopy and biotinylation experiments strongly suggest that only the very small fraction of METΔ7–8 that is cleaved reaches the cell membrane, but this fraction is insignificant with respect to the auto-active component in the cytosol.
From a genetic perspective, the METΔ7–8 mutant resembles the auto-active EGFRvIII variant which occurs concomitant with EGFR amplification in 25–64 % of all GBMs [51, 59]. This variant results from a genetic deletion of exons 2–7 and its activation is also independent of the ligands EGF or TNFα. Like the EGFRvIII mutation , the METΔ7–8 alteration appears to be restricted to only few cancer types, as we detected it in glioma but not in a number of other tumor types. The mutation was detected in grade III gliomas of both oligodendroglial and astrocytic origin and GBMs. Investigations of METΔ7–8 expression in other tumor types is however warranted and is ongoing in our lab.
Another example of a tyrosine kinase receptor which often carries deletions in GBM is PDGFRA. Forty percent of the GBMs with amplified PDGFRA contain an in-frame deletion in the ectodomain, leading to constitutive activation of the TK domain . Recently, also in pediatric high-grade gliomas, ligand-independent and tumorigenic in-frame PDGFRA deletion variants have been reported [47, 58]. Data on the subcellular localization of such mutated oncoreceptors are frequently lacking, and our data call for in-depth analysis of the cellular localization of receptors that are considered targetable. Interestingly, activating mutations in the RON tyrosine kinase receptor have been identified which resemble the METΔ7–8 mutation in that it also involves the first IPT domain . Of note, these alterations do not lead to a loss of expression at the cell surface.
Importantly, we did not detect a METwt transcript in E98 cells, in contrast to the clinical gliomas which were analyzed in this study and which all showed abundant wtMET, also in METΔ7–8 tumors. We formally cannot exclude that the wild-type MET amplicons in our PCR derive from ‘contaminating’ non-neoplastic stromal cells in the tumor biopsies that were tested. There may, however, be another explanation for the relatively low levels of METΔ7–8 in clinical tumors: in the patient tumor from which the E98 model was generated, a low percentage of MET-expressing tumor cells was detected, although it is impossible to determine whether these cells carry the METΔ7–8 mutation because the antibodies used do not discriminate between MET and METΔ7–8. It is tempting to speculate that during the generation of the E98 model, a small subset of METΔ7–8 tumor cells in the primary tumor experienced a growth advantage, ultimately resulting in clonal outgrowth during xenograft formation. This scenario fits with the notion that clinical tumors not only show inter- but also intratumoral heterogeneity , and derived preclinical tumor models may only be representative for the most malignant population of tumor cells.
Expression of METΔ7–8 may have important consequences for choice of therapy. MET is increasingly recognized as an important target in multiple tumor types, including glioma, and therapeutic antibodies against HGF or the HGF binding site on MET have been developed. Since HGF is not involved in METΔ7–8 activation and METΔ7–8 is retained intracellular, MET-mutated cells will not be responsive to these therapies. Indeed, we were able to show that E98 cells do not respond to the anti-MET VHH G2. Selection of MET-mutated cells in tumors that initially respond to antibody-based MET-directed treatment is expected to result in recurrence of treatment-resistant clones. In this respect, it will be important in future studies to assess the occurrence of the Δ7–8 mutation in paired samples of primary and recurrent tumors after MET antibody-based therapies, but also anti-EGFR therapies since cells may use METΔ7–8 to bypass EGFR signaling [3, 53]. With this in mind, the use of specific tyrosine kinase inhibitors of MET may have preference over antibody-based therapy for resistant tumors, at least in the ones that are KRAS and RAF wild type, [3, 21, 57, 61] since METΔ7–8 is sensitive to these inhibitors . Such inhibitors have already shown to improve overall survival of patients with non-small cell lung carcinoma with MET amplification and renal papillary carcinoma with MET mutations [11, 45]. A recent clinical study with anti-MET antibody MetMab for lung cancer failed to meet the primary endpoint of prolonged survival . A study of MET mutations and intracellular localization patterns in this patient group may be highly informative for future therapeutic directions .
In conclusion, we describe a highly active, non-ligand-dependent mutant of MET in 6 % of gliomas, which is not exposed on the cell surface and is predicted to be non-targetable with therapeutic antibodies against MET and/or HGF.
We thank I. Otte-Höller for assistance with confocal microscopy imaging, P.G. Gritsenko and J. Fransen for EEA1 and G1/296 antibodies, respectively, G. Röhn for help in preparing glioma cDNAs, J.T.G. Schepens for assistence with lentivirus generation and transductions, Y.M. Versleijen-Jonkers and M.H. Roeffen for providing cDNAs of a spectrum of sarcomas and F. Smit for providing CRPC cDNA. ACN was sponsored by an internal RUCO research grant. Semi-conductor sequencing was performed with financial support of Stichting STOPhersentumoren.nl.
Conflict of interest
The authors have no conflicts of interest to disclose.
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