Soluble Urokinase Receptor Is Released Selectively by Glioblastoma Cells That Express Epidermal Growth Factor Receptor Variant III and Promotes Tumor Cell Migration and Invasion

Genomic heterogeneity is characteristic of glioblastoma (GBM). In many GBMs, the EGF receptor gene (EGFR) is amplified and may be truncated to generate a constitutively active form of the receptor called EGFRvIII. EGFR gene amplification and EGFRvIII are associated with GBM progression, even when only a small fraction of the tumor cells express EGFRvIII. In this study, we show that EGFRvIII-positive GBM cells express significantly increased levels of cellular urokinase receptor (uPAR) and release increased amounts of soluble uPAR (suPAR). When mice were xenografted with human EGFRvIII-expressing GBM cells, tumor-derived suPAR was detected in the plasma, and the level was significantly increased compared with that detected in plasma samples from control mice xenografted with EGFRvIII-negative GBM cells. suPAR also was increased in plasma from patients with EGFRvIII-positive GBMs. Purified suPAR was biologically active when added to cultures of EGFRvIII-negative GBM cells, activating cell signaling and promoting cell migration and invasion. suPAR did not significantly stimulate cell signaling or migration of EGFRvIII-positive cells, probably because cell signaling was already substantially activated in these cells. The activities of suPAR were replicated by conditioned medium (CM) from EGFRvIII-positive GBM cells. When the CM was preincubated with uPAR-neutralizing antibody or when uPAR gene expression was silenced in cells used to prepare CM, the activity of the CM was significantly attenuated. These results suggest that suPAR may function as an important paracrine signaling factor in EGFRvIII-positive GBMs, inducing an aggressive phenotype in tumor cells that are EGFRvIII-negative.     

Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which the expressions of EGFR and EGFRvIII are maintained both in xenograft tumors growing subcutaneously on mice and in cell cultures established in stem cell conditions. With this model it will be possible to further study the role of EGFR and EGFRvIII, and response to targeted therapy, in GBM.     

Molecular Characterization of EGFR and EGFRvIII Signaling Networks in Human Glioblastoma Tumor Xenografts

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ∼50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.Glioblastoma multiforme (GBM)¹ is the most frequent and aggressive form of primary brain tumor (1). The current standard of care for GBM consists of surgical removal, radiotherapy, and adjuvant chemotherapy (typically temozolomide) (1). However, despite these interventions the prognosis is still poor, with mean survival time at ∼15 months following diagnosis (2). Genetic profiling of GBM tumors has been used to identify multiple distinct genetic aberrations across a diverse array of genes such as the deletion of phosphatase and tensin homolog (PTEN), p16 deletion, and mutation of TP53 (3, 4). Additionally, amplification, overexpression, and/or mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) has been identified to be a key genetic alteration in ∼50% of GBM patients (5). EGFR amplification is often accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors (6–8). EGFRvIII is characterized by the deletion of exon 2–7, resulting in an in-frame deletion of 267 amino acid residues from the extracellular domain. This deletion generates a receptor which is unable to bind ligand yet is constitutively, but weakly, active (9). Continuous low level activation leads to impaired internalization and degradation of the receptor, causing prolonged signaling (10). Expression of EGFRvIII in the absence of wtEGFR leads to the transformation of cells in vivo, drives cell proliferation in vitro, and expression of EGFRvIII correlates with poor prognosis in the clinic (6, 11, 12). EGFRvIII has been identified in GBM, lung, ovarian, and breast cancers, but has never been identified in normal tissue (13, 14). Because of the absence of this mutant receptor in normal tissue, EGFRvIII is an attractive therapeutic target. Although EGFR inhibitors, such as erlotinib and gefitinib, inhibit EGFR, EGFRvIII bearing xenograft models and cell lines are resistant to these inhibitors (15, 16). Therapeutic agents directly targeting EGFRvIII in murine GBM xenografts initially resulted in reduced tumor volume and a modest increase in survival (17). However, tumor recurrence was inevitable because of resistance by uncharacterized evasion mechanisms and adaptations (17). We propose that an improved understanding of the system-wide changes in protein expression and signaling caused by EGFRvIII expression should provide insight into specific therapeutic targets for EGFRvIII driven tumors.It is thought that EGFRvIIl enhances tumorigenicity by differential utilization (e.g. altered amplitude and kinetics and potentially novel components or pathways) of signal transduction pathways compared with ligand activated wtEGFR. Quantitative mass spectrometry has previously been applied to the identification of EGFRvIII specific phosphotyrosine signaling across four GBM cell lines expressing titrated levels of EGFRvIII relative to cells expressing the kinase-dead control (18). Cross-activation of EGFRvIII and the c-Met receptor tyrosine kinase is prevalent within these EGFRvIII overexpressing cell lines, revealing an attractive therapeutic strategy (18), which was later extended to include cross-activation of PDGFR (platelet-derived growth factor receptor) (19).Although EGFRvIII signaling has been extensively studied in GBM cell lines, the molecular mechanisms of increased tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated (20, 21). In addition, tissue culture conditions dramatically change the genetic and molecular characteristics found in primary human tumors. In particular, EGFRvIII expression is rapidly lost during generation of primary culture cells from GBM tumors. Most of the EGFRvIII-expressing cells lines are a result of stable transfection, rather than endogenous expression, of the mutant receptor (22). Additionally, the micro-environment and cellular heterogeneity of the tumor have a significant impact on the response to therapeutics, yet are poorly reflected in cell culture. As a consequence, quantification of signaling networks in glioblastoma cell lines provide a limited understanding of the signaling networks in GBM tumor samples.To overcome this limitation, the James and Sarkaria labs have generated, from patient surgical specimens, a panel of glioblastoma tumor xenografts that are maintained through serial passaging as subcutaneous xenografts in nude mice (22, 23). Maintenance of GBM tumors in this in vivo setting preserves the genetic features and phenotypes crucial to the tumorigenicity of the primary human tumors (23). With these tumor xenografts it is possible to analyze in vivo signaling networks, predict optimal therapeutic strategies based on these data, and test these predictions in a physiologically relevant system.To quantify signaling networks activated in glioblastoma tumor xenografts and determine the effect of wtEGFR or EGFRvIII expression on these networks, we applied quantitative mass spectrometry to eight human GBM xenografts expressing wtEGFR (wt) or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+) implanted into the flanks of nude mice. This analysis led to the identification and quantification of 1588 proteins (across two or more biological replicates) and 225 tyrosine phosphorylation sites on 168 proteins across eight tumor xenografts. Integration of quantitative phosphotyrosine data and protein expression profiles have uncovered the differential regulation of novel proteins and phosphotyrosine sites, which relate to the mode of action of wtEGFR and EGFRvIII overexpression in vivo. Quantification of tyrosine phosphorylation networks revealed signaling specific to each tumor xenograft. These data provide evidence for a significant amount of variation across the eight xenografts, and suggests that optimal therapeutic strategies might be specific to each tumor.     

The protein tyrosine phosphatase SHP-2 is required for EGFRvIII oncogenic transformation in human glioblastoma cells

Oncogenic EGFRvIII is a naturally occurring oncoprotein and is expressed in about 40-50% of human glioblastomas, particularly those that arise de novo. To understand the molecular mechanisms by which this oncoprotein alters transforming phenotypes, and since our previous work indicated that SHP-2 protein tyrosine phosphatase activity modulated EGFRvIII activation and downstream signaling, we examined whether SHP-2 plays a role in EGFRvIII-induced oncogenesis by using both PTEN-deficient U87MG.EGFRvIII and PTEN-intact LN229.EGFRvIII cells. Inhibition of SHP-2 expression by Shp-2 siRNA inhibited cell growth, transformation and altered morphology of these EGFRvIII transformed GBM cells. Ectopic expression of a PTPase-inactive form of SHP-2, SHP-2 C459S, but not its wild-type SHP-2 or either of two SH2 domain mutants, abrogated transformation of EGFRvIII-expressing glioblastomas in soft agar and in nude mice. SHP-2 C459S cells grew slower and exhibited a more flattened morphology with more organized actin stress fibers under both full growth and low serum conditions. Furthermore, shp-2+/− and −/− mouse embryonic fibroblasts (MEFs) could not be transformed by EGFRvIII while shp-2+/+ MEFs displayed a fully transformed phenotype upon introduction of EGFRvIII, again indicating a requirement for functional SHP-2 in EGFRvIII transformation. Moreover, the SHP-2 PTPase activity inhibitor NSC-87877 inhibited endogenous SHP-2 activity, Erk phosphorylation and transformation in both GBM cell lines. EGFRvIII expression recruited SHP-2 to the receptor complex to transduce signals and also increased SHP-2 phosphorylation at Tyr542. Inhibition of EGFRvIII-induced cell growth and transformation by SHP-2 C459S or shp-2 siRNA was mediated by its ability to block cell cycle progression at different phases in these GBM cells. These data indicate that differential activation of SHP-2 phosphorylation at Tyr542 in these two GBM cell lines likely results in increased different PTPase activity and distinct mechanisms of cell cycle progression and SHP-2, in particular its PTPase activity, plays a critical role in EGFRvIII-mediated transformation.     

Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition.

Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.     

A curcumin derivative hydrazinobenzoylcurcumin suppresses stem-like features of glioblastoma cells by targeting Ca2+/calmodulin-dependent protein kinase II

Glioblastoma multiforme (GBM) is the most aggressive and common type of human primary brain tumor. Glioblastoma stem-like cells (GSCs) have been proposed to contribute to tumor initiation, progression, recurrence, and therapeutic resistance of GBM. Therefore, targeting GSCs could be a promising strategy to treat this refractory cancer. Calmodulin (CaM), a major regulator of Ca²⁺-dependent signaling, controls various cellular functions via interaction with multiple target proteins. Here, we investigated the anticancer effect of hydrazinobenzoylcurcumin (HBC), a Ca ²⁺/CaM antagonist, against GSCs derived from U87MG and U373MG cells. HBC significantly inhibited not only the self-renewal capacity, such as cell growth and neurosphere formation but also the metastasis-promoting ability, such as migration and invasion of GSCs. HBC induced apoptosis of GSCs in a caspase-dependent manner. Notably, HBC repressed the phosphorylation of Ca ²⁺/CaM-dependent protein kinase II (CaMKII), c-Met, and its downstream signal transduction mediators, thereby reducing the expression levels of GSC markers, such as CD133, Nanog, Sox2, and Oct4. In addition, the knockdown of CaMKIIγ remarkably decreased the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking c-Met signaling pathway in U87MG GSCs. These results suggest that HBC suppresses the stem-like features of GBM cells via downregulation of CaM/CaMKII/c-Met axis and therefore CaMKII may be a novel therapeutic target to eliminate GSCs.     

In situ analysis of mutant EGFRs prevalent in glioblastoma multiforme reveals aberrant dimerization, activation, and differential response to anti-EGFR targeted therapy

Aberrations in epidermal growth factor receptor (EGFR/ErbB1) are the most common oncogenic alterations in glioblastoma multiforme (GBM), the most common primary brain tumor. Interactions between wild-type (wt) and mutant EGFRs and their subsequent activation are of biologic and potential therapeutic importance in GBMs. We recently showed that in situ proximity ligation assay (PLA) allows for quantitative evaluation of EGFR dimerization and activation in intact cells. Using this in situ platform, we show the aberrant homo-/heterodimeric properties of EGFRvIII and EGFRc958 mutants, the two most common EGFR mutants in GBMs. In addition, dimer phosphoactivation status could be detected by PLA with superior signal-noise ratio (>17-fold) and sensitivity (>16-fold) than immunofluorescence-based phospho-EGFR measurements. Dimer activation analysis indicated quantitative activation differences of mutant dimers. These aberrant features were not overexpression dependent but appeared independent of cellular expression levels, suggesting inherent properties of the mutant receptors. Moreover, we observed in situ detection of EGFRwt-EGFRvIII heterodimerization in GBM specimens, supporting our cell line observations. Notably, currently used anti-EGFR therapeutics, such as cetuximab, matuzumab, and panitumumab, could effectively block EGFRwt dimerization and activation but did not equally impair EGFRvIII homodimers, EGFRwt-EGFRvIII, or EGFRvIII-EGFRc958 heterodimers. EGFRvIII appears to have intrinsic phosphoactivation independent of dimerization as matuzumab blockade of homodimerization had no effect on receptor phosphorylation levels. These data suggest differences in the dimerization-blocking efficacy of EGFR monoclonal antibodies as mutant EGFR dimer configurations prevalent in GBMs can evade blockade by anti-EGFR treatments. Further studies are warranted to evaluate whether this evasion contributes to poor therapeutic response or resistance.     

Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.     

EGFRvIII-Specific Chimeric Antigen Receptor T Cells Migrate to and Kill Tumor Deposits Infiltrating the Brain Parenchyma in an Invasive Xenograft Model of Glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII⁺ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII⁺ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.     

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells

Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.     

Phosphoproteomic studies of receptor tyrosine kinases: future perspectives

In the last decade, large-scale mass spectrometry-based phosphoproteomic studies of receptor tyrosine kinases (RTKs) have generated a compendium of signalling networks that are activated downstream of these receptors. In this article, a brief summary of previous phosphoproteomic studies on epidermal growth factor receptor (EGFR) signalling will be presented together with a perspective on the importance for the field to keep pace with new advances in RTK biology. Using examples drawn primarily from studies on the EGFR, c-Met and Flt3 receptors, areas in RTK biology which will greatly benefit from the power of phosphoproteomics will be discussed, including (a) validating oncogenic RTK mutants identified in cancer genome sequencing efforts, (b) spatial RTK signalling networks and (c) understanding crosstalk and co-activation between members of the RTK superfamily.     

Development of an EGFRvIII specific recombinant antibody

Background  

EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.     

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.     

The effect of epidermal growth factor receptor variant III on glioma cell migration by stimulating ERK phosphorylation through the focal adhesion kinase signaling pathway

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.     

A new anti-c-met antibody selected by a mechanism-based dual-screening method: Therapeutic potential in cancer

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.     

Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors

Hepatocyte growth factor/scatter factor (HGF/SF) receptor c-Met is implicated in growth, invasion and metastasis of many tumors. Tumor cells harboring MET gene amplification are initially sensitive to c-Met tyrosine kinase inhibitors (TKI), but escape from long-term treatment has not been investigated. C-Met is a client of heat shock protein 90 (Hsp90) and is destabilized by Hsp90 inhibitors, suggesting that these drugs may inhibit tumors driven by MET amplification, although tumor escape under these conditions also has not been explored. Here, we evaluated the initial inhibitory effects of, and the likelihood of escape from, the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and the c-Met TKI SU11274, using two cell lines harboring MET gene amplification. 17-AAG inhibited cell growth in both cell lines and induced substantial apoptosis, whereas SU11274 was only growth inhibitory in one cell line. In both cell lines, c-Met-dependent Akt, Erk and/or STAT3 signaling, as well as activation of the EGFR family, resumed shortly after treatment with c-Met TKI despite sustained c-Met inhibition. PKC δ upregulation may participate in reactivation of c-Met downstream signaling in both cell lines. In contrast to c-Met TKI, 17-AAG destabilized c-Met protein and durably blocked reactivation of downstream signaling pathways and EGFR family members. Our data demonstrate that downstream signaling in tumor cells over-expressing c-Met is not stably suppressed by c-Met TKI, even though c-Met remains fully inhibited. In contrast, Hsp90 inhibitors provide long-lasting suppression of c-Met-dependent signaling, and these drugs should be further evaluated in tumors driven by MET gene amplification.