Abstract
Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor–c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.
Introduction
Glioblastoma is the most malignant central nervous system cancer and accounts for the majority of primary brain cancer–related deaths (Porter et al., 2010). Despite advances in multimodality therapies, such as surgery, radiotherapy, and chemotherapy, the outcome for patients remains extremely poor, with an average postdiagnostic survival of just over 14 months (Behin et al., 2003; Stupp et al., 2005; Louis et al., 2007). The high mortality rate results from the universal resurgence of tumors post-treatment, which occurs due to infiltrating tumor cells that escape initial surgery and exhibit profound resistance to irradiation and current chemotherapy treatments (Claes et al., 2007). Thus, identification of novel tractable targets for improved therapeutics is desperately needed.
Genomic and proteomic analyses have identified a number of key oncogenic drivers of glioblastoma multiforme (GBM) tumorigenesis and therapeutic resistance, including receptor tyrosine kinases (RTKs) (Beroukhim et al., 2007; Huang et al., 2007a,b). In particular, amplification of the epidermal growth factor receptor (EGFR) is present in approximately half of all GBMs (Hurtt et al., 1992; Jaros et al., 1992), with a large proportion also expressing activating mutations, such as deletion of exons 2–7, which results in a ligand-independent, constitutively active mutant commonly referred to as EGFR variant III (EGFRvIII) (Nishikawa et al., 1994; Batra et al., 1995; Huang et al., 1997). While it is unclear how EGFRvIII mutations are generated, it appears to occur late during tumor progression and its coexpression with the wild-type receptor have been reported to confer poor prognosis and a shorter patient survival time (Shinojima et al., 2003; Heimberger et al., 2005). This phenomenon is also observed in orthotopic xenograft mouse studies, whereby expression of EGFRvIII in human GBM cell lines leads to an overwhelming enhancement in tumor growth, invasion, and resistance to radiation and chemotherapies (Nagane et al., 1996; Huang et al., 1997).
The c-Jun NH2-terminal family of kinases (JNK) consists of three isoforms, JNK1, JNK2, and JNK3, which are activated by a variety of stimuli, including UV light, cytokines, and growth factor signaling (Davis, 2000). JNKs have many cellular substrates and play diverse cellular roles, from induction of apoptosis to proliferation, depending upon the cell type and initial stressor (Kennedy and Davis, 2003). In GBM, constitutive JNK activation is observed in up to 86% of human glioblastomas, where activation strongly correlates with both histologic grade and expression of EGFR (Antonyak et al., 2002; Cui et al., 2006; Li et al., 2008). JNK1 and JNK2 are further known to regulate GBM stem cell–like characteristics and tumor initiating potential. Most notably, genetic knockdown of JNK2 impairs intracranial tumor formation and extends survival in GBM mouse models, suggesting that this particular isoform is important for the pathology of the disease (Matsuda et al., 2012; Yoon et al., 2012).
Here, we have investigated the involvement of the JNK2 isoform in EGFRvIII-driven GBM tumorigenesis. We demonstrate that JNK2 is necessary for the growth of aggressive EGFRvIII-positive tumors in vivo and invasiveness ex vivo. Our mechanistic studies identify a critical role for the EGFRvIII-JNK2 signaling axis in the regulation of HGF production and c-Met activation. Importantly, JNK2-dependent secretion of HGF is sufficient to activate c-Met, both in a cell intrinsic and extrinsic manner, where conditioned media from EGFRvIII-expressing cells promotes c-Met activation in cells that do not express the mutated receptor. Finally, we demonstrate functional importance of this signaling circuit in the promotion of cellular invasion. The addition of exogenous hepatocyte growth factor (HGF) is sufficient to partially restore invasion of JNK2-depleted cells. Together, these data define a critical role for JNK2 in the activation of a HGF/c-Met signaling downstream of EGFRvIII in GBM.
Materials and Methods
Cell Lines and Reagents.
U87 and T98G human glioblastoma cells were purchased from the American Type Culture Collection (Manassas, VA). DK-MG cells were purchased from Leibniz-Institut DSMZ (Braunschweig, Germany). Modified U87 cell lines were a kind gift from Dr. Frank Furnari (Ludwig Institute for Cancer Research, University of California at San Diego, San Diego, CA). GBM6 cells were a kind gift from Dr. Jann N. Sarkaria (Department of Radiation Oncology, Mayo Clinic, MN). GBM6 cells were maintained by serial passage in the flanks of nude mice and cultured short term as described previously (Carlson et al., 2011). Cell lines were maintained in Dulbecco’s minimum essential medium (DMEM) with glutamax (GIBCO by Life Technologies, Grand Isle, NY) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO). Cells were incubated at 37°C, 5% CO2, and 95% humidity. U87vIII and U87vIII-KD cells were maintained in media containing G418 (Life Technologies). To study the effects of conditioned media (CM), cells were seeded for 24 hours and subsequently serum starved for an additional 24 hours. CM was obtained from cells in serum-free media and clarified by centrifugation.
Lentiviral Transduction.
Sequence-specific small hairpin RNA (shRNA) pGIPZ vectors for the inhibition of JNK2 or nonsilencing control were purchased from Open Biosystems (Thermo Fischer Scientific, Pittsburg, PA). shRNA JNK2 sequences were 1) 5′-CTAGCAACATTGTTGTGAA-3′; 2) 5′- CTTCTGAAGTTATCTCTTA-3′; and 3) 5′-GCATTAAAGCAGCGTATC-3′. Lentiviral particles were generated using the Trans-Lentiviral shRNA packaging kit (TLP5912; Thermo Fischer Scientific) per the manufacturer’s recommendations. U87vIII cells (1 × 106) were seeded in 10-cm2 dishes and transduced with shRNA lentiviruses for a period of 72 hours. Transduced cells were selected by the addition of 1 µg/ml of puromycin (Sigma). Stable cell lines were maintained at 37°C in a humidified 5% CO2 atmosphere in DMEM with glutamax (GIBCO by Life Technologies) supplemented with 10% FBS.
Western Blot Analysis.
GBM cell lines were seeded into six-well plates at a concentration of 2.5 × 105 cells/well, cell media was removed, and cells were washed in ice-cold phosphate-buffered saline and pelleted. Cells were lysed in 100 μl of ice-cold RIPA buffer (Boston BioProducts, Ashland, MA) containing protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN) and centrifuged at 14,000g for 15 minutes at 4°C. Protein concentrations were determined using the Pierce BCA protein assay reagent (Thermo Scientific, Rockford, IL). Samples were analyzed using NuPAGE Novex Bis-Tris polyacrylamide gel electrophoresis (Life Technologies). Gels containing the separated protein were transferred to nitrocellulose membranes using standard protocols. Membranes were probed with the following antibodies: anti-EGFR, anti–phospho-EGFR (Y1068), anti–c-Met, anti–phospho-c-Met (Y1234/1235), anti–phospho-c-Met (Y1349), anti–phospho-GRB2-associated binding protein 1 (Y307), anti-JNK2, anti–glyceraldehyde 3-phosphate dehydrogenase, or anti–α-tubulin antibodies. All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA) and used at a 1:1000 dilution. After repeated washes with Tris-buffered saline with 0.1% Tween 20 (20 mM Tris, pH 7.6, 140 mM NaCl, and 0.1% Tween 20), membranes were incubated with the appropriate IRDye-conjugated secondary antibody (LI-COR Biosciences, Lincoln, NE) in a 1:10,000 dilution. Membranes were imaged using the LI-COR Odyssey infrared imaging system (LI-COR Biosciences). Human HGF polyclonal antibody was purchased from R&D Systems (Minneapolis, MN).
Enzyme-Linked Immunosorbent Assay.
Cells (2.5 × 105) were plated in six-well plates for 24 hours and subsequently serum starved for an additional 24 hours. Human hepatocyte growth factor levels were determined using a human HGF enzyme–linked immunosorbent assay kit per the manufacturer’s recommendation (Invitrogen by Life Technologies).
Invasion Assays.
Invasion assays were performed using the CultureCoat 24-well basement membrane extract–coated cell invasion assay (Trevigen Inc., Gaithersburg, MD). The assay consist of an 8-µm pore sized modified Boyden chamber coated with a thin layer of basement membrane extract. Cells were cultured to 80% confluence and subsequently serum starved for 4–6 hours, resuspended in serum-free media, and added to the top of each chamber per the manufacturer’s recommendations. Recombinant human HGF (40 ng/ml) was added to the cells (R&D Systems). DMEM with glutamax supplemented with 10% FBS was added to the bottom of each chamber (500 µl). Invasive cells on the underside of the membranes were dissociated in a solution containing calcein-AM. The plates were analyzed using a SpectraMax (Molecular Devices, Sunnyvale, CA) plate reader, with fixed excitation and emission wavelengths (485/520 nm).
Microarray Analysis.
Total mRNA was extracted from GBM cell lines using the RNeasy Plus Mini Kit (Qiagen Sciences, Germantown, MD). Using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), RNA (10 μg) was reverse transcribed into single-stranded cDNA per the manufacturer’s recommendations. Samples were heated to 95°C for ∼1 minute to denature RNA/cDNA hybrids, followed by treatment with RNaseA for 30 minutes at 37°C, and DNA was purified using a QIAquick PCR Purification Kit (Qiagen Sciences) per the manufacturer’s recommendation. DNA concentrations were calculated using the NanoDrop 1000 spectrophotometer (Thermo Scientific). Hybridization of the cDNA to the Human Gene Expression 12x135K array was performed at the Florida State University NimbleGen Microarray Facility. Differentially expressed genes were identified using the following criteria: fold change > 2 in both directions and unpaired t test P value < 0.05. Three hundred and sixty probes were identified as the “JNK2 signature” and were imported into the Ingenuity pathway analysis tool (IPA) for pathway and upstream regulator analysis.
In Vivo Tumor Xenograft.
Male Nu/Nu mice were obtained from Charles River Laboratories (Wilmington, MA), received food and water ad libitum, and were kept in a controlled environment at an ambient temperature on a 12-hour light/dark cycle. Experiments were performed when animals reached 5–6 weeks of age, upon which mice were subcutaneously inoculated in the flank with 7.5 × 105 GBM cells. Tumors were measured using calipers, and tumor volumes were determined using methodologies where width, length, and height are used [e.g., v = (π/6) × (L × W × H)] (Tomayko and Reynolds, 1989). All animal studies were approved by the Scripps-Florida Institutional Animal Care and Use Committee, and care and maintenance were in accordance with the principles described in the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication 85-23, 1985).
Statistical Analysis.
All values in the figures are presented as the mean ± standard deviation of at least three independent experiments, except for the in vivo study, where the values are presented as the mean ± standard error of the mean. All the experiments were analyzed using a Student’s t test, where significant P values were presented as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
Results
JNK2 Is Required for EGFRvIII-Driven GBM Tumorigenicity.
To investigate the role of JNK2 in EGFRvIII-mediated GBM tumorigeneis, we first examined JNK2 pathway activation in GBM cells expressing EGFRvIII. U87 cells were engineered to express the mutated receptor and analyzed for activated signaling by measuring phosphorylation of the downstream transcription factor c-Jun. Using this approach, we identified that EGFRvIII expression in both U87 cells as well as the patient-derived line GBM6 resulted in the activation of JNK2. In contrast, phosphorylation of the downstream transcription factor c-Jun was not detected in U87 cells in the absence of the mutant EGFR, and silencing of JNK2 was sufficient to impair activation of c-Jun (Fig. 1A). These findings suggest an important role for the JNK2 isoform in the activation of signaling downstream of EGFRvIII. Since previous studies have identified a role for JNK2 in the promotion of a tumorigenic phenotype in GBM (Antonyak et al., 2002; Cui et al., 2006), we examined the effects of JNK2 shRNAs on the growth of U87vIII cells in vivo. JNK2 knockdown resulted in a significant reduction in tumor growth over time when compared with either nontransduced cells or those expressing nontargeting shRNAs (Fig. 1B).
JNK2 Mediates EGFRvIII-Induced Cellular Invasion.
To elucidate the molecular mechanisms by which JNK2 confers tumorigenicity to U87vIII, cells expressing either nontargeting or JNK2 shRNAs were subjected to microarray analysis. Using this approach, we identified 360 differentially expressed genes (P ≤ 0.05; >2-fold change), with the majority of transcripts showing decreased expression upon JNK2 knockdown (Fig. 1C). IPA further revealed key canonical pathways regulated by JNK2, which included categories, such as cancer, tissue development, cellular growth and proliferation, and inflammation (Fig. 1D). Notably, the most significantly enriched category was cellular movement, with 68 genes related to this pathway showing differential expression upon JNK2 knockdown (Supplemental Table 1). These observations are consistent with a large body of evidence showing that EGFRvIII mediates the invasiveness of GBM tumors (Zhu et al., 2009; Dunn et al., 2012) and implicates a role for JNK2 in this phenotype. We therefore examined the effects of JNK2 knockdown on cellular invasion. Since JNK2 is implicated in GBM cell proliferation and to distinguish this effect from the invasive phenotype, all the experiments were performed in a short-term period after modulation of JNK2 expression where the proliferation was not modulated. As expected, EGFRvIII-expressing U87 cells showed an almost 2-fold increase in invasive capacity using a Boyden chamber assay. Further, silencing of JNK2 in U87 and DK-MG EGFRvIII-expressing cells resulted in a significant reduction in invasion through the basement membrane coated transwell (Fig. 1, E and F), suggesting that JNK2 is indeed an important mediator of GBM invasiveness in EGFRvIII-expressing cells.
JNK2 Is Required for HGF Production in EGFRvIII-Expressing GBM Cells.
To identify key effectors of JNK2-mediated invasiveness, we performed further pathway analyses on our microarray data. Using the IPA software, we evaluated known gene interactions within the signature list (Fig. 2, A and B). A number of genes whose products are known to be associated with cellular movement were identified as either direct or indirect signaling mediators, including HGF, insulin-like growth factor binding protein 5, matrix metallopeptidase 7, platelet derived growth factor receptor alpha, and signal transducer and activator of transcription 5A (Fig. 2A; Supplemental Table 1). Importantly, HGF was identified as the most significantly predicted upstream regulator of the gene signature associated with JNK2 knockdown (Fig. 2B) (IPA overlap P value = 5.72 × 105). HGF is the only known activating ligand for the RTK c-Met (Lai et al., 2009) and is known to have tumor-promoting roles in GBM, including promotion of cell proliferation, cell migration, and maintenance of self-renewal, leading to therapeutic resistance of glioma stem cells (Joo et al., 2012).
Like JNK2, increased HGF expression and c-Met activation is associated with an advanced tumor grade and poor prognosis in patients with GBM (Laterra et al., 1997; Lamszus et al., 1999). Accordingly, the treatment of GBM xenografts with either neutralizing anti-HGF antibody or c-Met kinase inhibitor (crizotinib) impairs glial tumor growth (Li et al., 2005; Kim et al., 2006; Martens et al., 2006; Rath et al., 2013). Moreover, studies indicate that constitutive EGFR/EGFRvIII signaling results in transactivation of additional RTKs, such as platelet derived growth factor receptor, vascular endothelial growth factor receptor, as well as c-Met itself (Huang et al., 2007b; Stommel et al., 2007). Based on these findings, we hypothesized that JNK2 is required for the activation of HGF/c-MET signaling in EGFRvIII-expressing GBM cells. To investigate this, we first examined HGF production in U87 cells, U87 cells expressing EGFRvIII, and those with a kinase dead version of the mutant receptor. Notably, EGFRvIII kinase activity resulted in an induction of HGF in these cells, as measured by enzyme-linked immunosorbent assay (Fig. 2C). Further, activation of c-Met (p–c-Met) and its downstream adaptor molecule GRB2-associated binding protein 1 was observed upon expression of active EGFRvIII in U87 and T98G cells (Fig. 2, D and E) (Li et al., 2013). In the case of the kinase dead mutant, we observed a slight increase in p–c-Met (Y1234/1235) levels, but no increase in p–c-Met (Y1345) or activation of downstream adaptor molecule GRB2-associated binding protein levels and HGF levels were significantly lower compared with active EGFRvIII. Kinase independent roles for EGFR have been observed and may explain the slight increase in p–c-Met (Y1234/1235) levels (Zhu et al., 2010; Tan et al., 2015). We next investigated the requirement for JNK2 in this process by studying the effects of JNK2 shRNAs on both HGF production and activation of c-Met signaling. In confirmation of our gene expression data, knockdown of JNK2 reduced HGF expression in U87 cells expressing EGFRvIII (Fig. 2F). Moreover, JNK2 loss resulted in impaired activation of both c-Met and GRB2-associated binding protein 1 in these cells as well as in DK-MG EGFRvIII–expressing cells, demonstrating a critical role for JNK2 in EGFRvIII-mediated activation of c-Met (Fig. 2, G and H).
EGFRvIII/JNK2–Induced HGF Expression Mediates Cellular Crosstalk.
Expression of EGFRvIII in glioblastoma is heterogeneous and usually observed in a subpopulation of tumor cells (Nishikawa et al., 2004). Indeed, EGFRvIII-expressing tumors rarely arise independently of amplified EGFR, and experimental models imitating the human condition have revealed that tumors containing a small fraction of EGFRvIII-expressing cells significantly enhance tumor growth, reducing overall survival (Nagane et al., 1996; Inda et al., 2010). Interleukin 6 and leukemia inhibitory factor, for example, were identified as EGFRvIII-induced paracrine factors that stimulated tumorigenicity (Inda et al., 2010). To this end, we hypothesized that JNK2 mediated production of HGF by EGFRvIII-expressing GBM cells may serve to transactivate c-Met signaling in non–EGFRvIII-expressing tumor cells. To test this, CM derived from U87 cells expressing either EGFRvIII or the kinase dead mutant was first added to U87 cells and activation of c-Met was examined. CM derived from EGFRvIII-expressing cells significantly induced phospho–c-Met in contrast to U87 cells grown in normal media. Further, the phospho–c-Met level was markedly reduced in cells incubated with CM derived from EGFRvIII-kinase dead expressing cells (Fig. 3, A and B). To confirm that HGF is the active component of the EGFRvIII CM, we used a neutralizing antibody that efficiently blocks activation of c-Met in T98G cells treated with recombinant human HGF (Supplemental Fig. 1). Pretreatment of U87vIII CM with the HGF antibody impaired activation of c-Met in T98G target cells (Fig. 3C). Consistent with this, transfection of U87vIII cells with small interfering RNAs directed against HGF prior to collection of CM also abrogated activation of c-Met in T98G cells, demonstrating a critical role for HGF in the activation of c-Met by EGFRvIII (Fig. 3D) (Huang et al., 2007b).
We next assessed whether inhibition of JNK2 can block the HGF/c-Met paracrine signaling in GBM cells. Conditioned media was isolated from cultures of U87, U87vIII, U87vIII-shNT, or U87vIII-shJNK2 cells and incubated with T98G cells. Cells treated with CM derived from JNK2 knockdown cells resulted in a significant reduction in phospho–c-Met levels compared with those treated with the CM of cells expressing a nontargeting shRNA (Fig. 4A). Moreover, exposure of these cells to CM derived from U87vIII cells pretreated with the pan-JNK inhibitor SP600125 (due to a lack of isoform-specific inhibitor) significantly reduced the activation of c-Met (Fig. 4B). Finally, we addressed whether the role of JNK2 in cellular invasion is dependent on the EGFRvIII-HGF signaling circuit. The addition of recombinant human HGF to invading cultures was able to partially restore the invasive capacity of U87 EGFRvIII-JNK2 knockdown cells (Fig. 4C). Moreover, the addition of recombinant human HGF did not rescue the growth inhibition of the U87 EGFRvIII shJNK2 cells (Supplemental Fig. 2). This suggests that while the EGFRvIII-JNK2 axis plays a role in GBM cell proliferation, this is driven through a pathway independent of HGF/c-Met. Collectively, our findings elucidate critical signaling interactions between EGFRvIII-JNK2 and the HGF–c-Met pathway in GBM cell invasion (Fig. 5).
Discussion
Major clinical challenges in the treatment of GBM include the ability of infiltrating tumor cells to disperse into distant brain tissue and the refractory nature of these cells to current therapies (Lefranc et al., 2005; Claes et al., 2007). Accordingly, identification of oncogenic signaling pathways driving GBM invasiveness and disease progression is required to develop targeted therapies, leading to prolonged overall survival. GBMs are, however, known to be highly heterogeneous in terms of their molecular profiling, with complex signaling interactions governing differential responses to therapeutic intervention. For example, several protumorigenic mechanisms have been linked to signaling via EGFRvIII, the constitutively active EGFR deletion mutant common to GBM (Huang et al., 2007b; Stommel et al., 2007; Bonavia et al., 2012). EGFR signaling is a known driver of GBM pathogenesis, where it has been shown to promote tumor initiation and development as well as infiltration of surrounding brain tissue and resistance to therapy. Importantly, EGFRvIII is able to transactivate multiple RTKs within the same tumor, resulting in overlapping and redundant signaling pathways (Huang et al., 2007b; Stommel et al., 2007). For example, c-Met is activated by EGFRvIII in GBM, where it is associated with advanced tumor grade and poor prognosis (Laterra et al., 1997; Lamszus et al., 1999). Importantly, such complexity is thought to account for the lack of significant efficacy of EGFR-targeted agents in the clinic (Taylor et al., 2012), with recent studies demonstrating that combined inhibition of EGFRvIII and c-Met synergistically impairs tumor cell growth in mouse models of GBM and lung cancer (Huang et al., 2007b; Lai et al., 2009; Lal et al., 2009; Pillay et al., 2009). These findings suggest that an understanding of the complex signaling interactions in human cancers are required for the rational design of combined and effective targeted therapeutic strategies.
Amplification of c-Met and HGF has been associated with highly invasive and metastatic tumors and poor prognosis in multiple tumors types. In GBM, EGFRvIII overexpression is known to drive activation of c-Met via a previously uncharacterized mechanism (Huang et al., 2007b). In this study, we define a role for JNK2 as a central mediator of the EGFRvIII-HGF/c-Met signaling circuit in GBM. Specifically, EGFRvIII-dependent activation of JNK2 is required for increased cellular invasion through transcriptional upregulation of a cast of genes involved in tumor cell movement, including HGF. We show that JNK2-dependent secretion of HGF leads to activation of c-Met in EGFRvIII-expressing cells, both in a paracrine- and autocrine-dependent manner. Phospho–c-Met is observed not only in GBM cells expressing the mutated receptor, but also in cells that lack EGFRvIII expression following incubation with conditioned media isolated from EGFRvIII-expressing cells. Confirmation of a role for secreted HGF ligand EGFRvIII cellular crosstalk was achieved using neutralizing antibodies and small interfering RNAs targeted against HGF. Finally, we established that JNK2-HGF signaling is important for GBM cellular invasion and recombinant HGF rescues the inhibitory effects of JNK2 knockdown. Collectively, our findings elucidate critical signaling interactions in GBM cell invasiveness and cellular crosstalk, both of which are important pathologic features of GBM. Further, our findings suggest that selective inhibitors of JNK2 could represent potential therapeutics for use in GBM, particularly when combined with additional RTK inhibitors, including those targeted against EGFR and c-Met.
Acknowledgments
We sincerely thank Ms. Pamela Clark-Spruill for secretarial assistance and members of the Duckett Laboratory for input and editing of the manuscript. This work was supported in part by a grant from the Florida Center for Brain Tumor Research, Accelerate Brain Cancer Cure, the Rendina Family Foundation, and funds from the State of Florida to Scripps Florida.
Authorshipship Contributions
Participated in research design: Saunders, Lafitte, Adrados, Quereda, Feurstein, Duckett.
Conducted experiments: Saunders, Lafitte, Adrados, Quereda, Ling.
Contributed new reagents or analytic tools: Fallahi.
Performed data analysis: Saunders, Lafitte, Fallahi, Adrados, Quereda, Ling, Rosenberg, Duckett.
Wrote or contributed to the writing of the manuscript: Saunders, Lafitte, Rosenberg, Adrados, Quereda, Fallahi, Duckett.
Footnotes
- Received January 7, 2015.
- Accepted September 30, 2015.
↵1 V.C.S. and M.L. contributed equally to this work.
This work is supported by The Rendina Family Foundation, 2011–2013 Florida Center for Brain Tumor Research (FCBTR)/Accelerate Brain Cancer Cure (ABC2) Grant, and funds from the State of Florida to Scripps Florida. The authors declare no competing financial interests.
↵This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- CM
- conditioned media
- DMEM
- Dulbecco’s minimum essential medium
- EGFR
- epidermal growth factor receptor
- EGFRvIII
- epidermal growth factor receptor variant III
- FBS
- fetal bovine serum
- GBM
- glioblastoma multiforme
- HGF
- hepatocyte growth factor
- IPA
- ingenuity pathway analysis
- JNK2
- c-Jun N-terminal kinase isoform 2
- p–c-Met
- activation of c-Met
- RTK
- receptor tyrosine kinase
- shRNA
- small hairpin RNA
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
References
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