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.     

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.     

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.     

Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility

EphA2 overexpression has been reported in many cancers and is believed to play an important role in tumor metastasis and angiogenesis. We show that the activated epidermal growth factor receptor (EGFR) and the cancer-specific constitutively active EGFR type III deletion mutant (EGFRvIII) induce the expression of EphA2 in mammalian cell lines, including the human cancer cell lines A431 and HN5. The regulation is partially dependent on downstream activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and is a direct effect on the EphA2 promoter. Furthermore, EGFR and EphA2 both localize to the plasma membrane and EphA2 coimmunoprecipitates with activated EGFR and EGFRvIII. Ligand activation of EphA2 and EphA2 knockdown by small interfering RNA inhibit EGF-induced cell motility of EGFR-overexpressing human cancer cells, indicating a functional role of EphA2 in EGFR-expressing cancer cells.     

Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects β-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.     

Cell adhesion and EGFR activation regulate EphA2 expression in cancer

EphA2 is frequently overexpressed in cancer, and increasing amounts of evidence show that EphA2 contributes to multiple aspects of the malignant character including angiogenesis and metastasis. Several aspects of the regulation and functional significance of EphA2 expression in cancer are still largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability. These effects were however abolished by activation of the EGF-receptor ligand system favoring Ras/MAPK signaling and cell proliferation. Based on our results, we propose a regulatory mechanism where cell adhesion induces EGFR kinase activation and EphA2 expression; and where the effect of ephrinA1 mediated reduction in cell viability by inhibiting EphA2 expression is overruled by activated EGFR in human cancer cells.     

Uncovering Therapeutic Targets FOR Glioblastoma: A Systems Biology Approach

Even though glioblastoma, WHO grade IV (GBM) is one of the most devastating adult cancers, current treatment regimens have not led to any improvements in patient life expectancy or quality of life. The constitutively active EGFRvIII receptor is one of the most commonly mutated proteins in GBM and has been linked to radiation and chemotherapeutic resistance. To define the mechanisms by which this protein alters cell physiology, we have recently performed a phosphoproteomic analysis of EGFRvIII signaling networks in GBM cells. The results of this study provided important insights into the biology of this mutated receptor, including oncogene dose effects and differential utilization of signaling pathways. Moreover, clustering of the phosphoproteomic data set revealed a previously undescribed crosstalk between EGFRvIII and the c-Met receptor. Treatment of the cells with a combination employing both EGFR and c-Met kinase inhibitors dramatically decreased cell viability in vitro. In this perspective, we highlight the use of systems biology as a tool to better understand the molecular basis of GBM tumor biology as well as to uncover non-intuitive candidates for therapeutic target validation.     

A novel extracellular Hsp90 mediated co-receptor function for LRP1 regulates EphA2 dependent glioblastoma cell invasion

Background

Extracellular Hsp90 protein (eHsp90) potentiates cancer cell motility and invasion through a poorly understood mechanism involving ligand mediated function with its cognate receptor LRP1. Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal brain cancers. The receptor tyrosine kinase EphA2 is overexpressed in the majority of GBM specimens and is a critical mediator of GBM invasiveness through its AKT dependent activation of EphA2 at S897 (P-EphA2_S897). We explored whether eHsp90 may confer invasive properties to GBM via regulation of EphA2 mediated signaling.

Principal Findings

We find that eHsp90 signaling is essential for sustaining AKT activation, P-EphA2_S897, lamellipodia formation, and concomitant GBM cell motility and invasion. Furthermore, eHsp90 promotes the recruitment of LRP1 to EphA2 in an AKT dependent manner. A finding supported by biochemical methodology and the dual expression of LRP1 and P-EphA2_S897 in primary and recurrent GBM tumor specimens. Moreover, hypoxia mediated facilitation of GBM motility and invasion is dependent upon eHsp90-LRP1 signaling. Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.

Significance

We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function. We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2. Taken together, our results demonstrate activation of the eHsp90-LRP1 signaling axis as an obligate step in the initiation and maintenance of AKT signaling and EphA2 activation, thereby implicating this pathway as an integral component contributing to the aggressive nature of GBM.     

Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

Background

Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting.

Results

A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants.

Conclusions

Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome.     

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.     

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D₃ receptor (D₃R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D₃R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D₃R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D₃R, which can be extended to the crosstalk between other RTKs and GPCRs.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual EGFR/HER2 Tyrosine Kinase Inhibitors

4-Anilinoquinazolines as an important class of protein kinase inhibitor are widely investigated for epidermal growth factor receptor (EGFR) tyrosine kinase or epidermal growth factor receptor 2 (HER2) inhibition. A series of novel 6-salicyl-4-anilinoquinazoline derivatives 9–27 were prepared and evaluated for their EGFR/HER2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on three variant cancer cell lines (A431, MCF-7, and A549). The bioassay results showed most of the designed compounds exhibited moderate to potent in vitro inhibitory activity in the enzymatic and cellular assays, of which compound 21 revealed the most potent dual EGFR/HER2 inhibitory activity, with IC₅₀ values of 0.12 µM and 0.096 µM, respectively, comparable to the control compounds Erlotinib and Lapatinib. Furthermore, the kinase selectivity profile of 21 was accessed and demonstrated its good selectivity over the majority of the close kinase targets. Docking simulation was performed to position compound 21 into the EGFR/HER2 active site to determine the probable binding pose. These new findings along with molecular docking observations could provide an important basis for further development of compound 21 as a potent EGFR/HER2 dual kinase inhibitor.     

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.     

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.     

The contribution of tumor and host tissue factor expression to oncogene-driven gliomagenesis

Glioblastoma multiforme (GBM) is an aggressive form of glial brain tumors, associated with angiogenesis, thrombosis, and upregulation of tissue factor (TF), the key cellular trigger of coagulation and signaling. Since TF is upregulated by oncogenic mutations occurring in different subsets of human brain tumors we investigated whether TF contributes to tumourigenesis driven by oncogenic activation of EGFR (EGFRvIII) and RAS pathways in the brain. Here we show that TF expression correlates with poor prognosis in glioma, but not in GBM. In situ, the TF protein expression is heterogeneously expressed in adult and pediatric gliomas. GBM cells harboring EGFRvIII (U373vIII) grow aggressively as xenografts in SCID mice and their progression is delayed by administration of monoclonal antibodies blocking coagulant (CNTO 859) and signaling (10H10) effects of TF in vivo. Mice in which TF gene is disrupted in the neuroectodermal lineage exhibit delayed progression of spontaneous brain tumors driven by oncogenic N-ras and SV40 large T antigen (SV40LT) expressed under the control of sleeping beauty transposase. Reduced host TF levels in low-TF/SCID hypomorphic mice mitigated growth of glioma subcutaneously but not in the brain. Thus, we suggest that tumor-associated TF may serve as therapeutic target in the context of oncogene-driven disease progression in a subset of glioma.     

A New EGFR Inhibitor from Ficus benghalensis Exerted Potential Anti-Inflammatory Activity via Akt/PI3K Pathway Inhibition

Inflammation is a critical defensive mechanism mainly arising due to the production of prostaglandins via cyclooxygenase enzymes. This study aimed to examine the anti-inflammatory activity of fatty acid glucoside (FAG), which is isolated from Ficus benghalensis against lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The cytotoxic activity of the FAG on RAW 264.7 macrophages was evaluated with an MTT assay. The levels of PGE2 and NO and the activity of iNOS, COX-1, and COX-2 enzymes in LPS-stimulated RAW 264.7 cells were evaluated. The gene expression of IL-6, TNF-α, and PGE2 was investigated by qRT-PCR. The expression of epidermal growth factor receptor (EGFR), Akt, and PI3K proteins was examined using Western blotting analysis. Furthermore, molecular docking of the new FAG against EGFR was investigated. A non-cytotoxic concentration of FAG increased NO release and iNOS activity, inhibited COX-1 and COX-2 activities, and reduced PGE2 levels in LPS-stimulated RAW 264.7 cells. It diminished the expression of TNF-α, IL-6, PGE2, EGFR, Akt, and PI3K. Furthermore, the molecular docking study proposed the potential direct binding of FAG with EGFR with a high affinity. This study showed that FAG is a natural EGFR inhibitor, NO-releasing, and COX-inhibiting anti-inflammatory agent via EGFR/Akt/PI3K pathway inhibition.