Loss of the tyrosine phosphatase PTPRD leads to aberrant STAT3 activation and promotes gliomagenesis

PTPRD, which encodes the protein tyrosine phosphatase receptor-δ, is one of the most frequently inactivated genes across human cancers, including glioblastoma multiforme (GBM). PTPRD undergoes both deletion and mutation in cancers, with copy number loss comprising the primary mode of inactivation in GBM. However, it is unknown whether loss of PTPRD promotes tumorigenesis in vivo, and the mechanistic basis of PTPRD function in tumors is unclear. Here, using genomic analysis and a glioma mouse model, we demonstrate that loss of Ptprd accelerates tumor formation and define the oncogenic context in which Ptprd loss acts. Specifically, we show that in human GBMs, heterozygous loss of PTPRD is the predominant type of lesion and that loss of PTPRD and the CDKN2A/p16^INK4A tumor suppressor frequently co-occur. Accordingly, heterozygous loss of Ptprd cooperates with p16 deletion to drive gliomagenesis in mice. Moreover, loss of the Ptprd phosphatase resulted in phospho-Stat3 accumulation and constitutive activation of Stat3-driven genetic programs. Surprisingly, the consequences of Ptprd loss are maximal in the heterozygous state, demonstrating a tight dependence on gene dosage. Ptprd loss did not increase cell proliferation but rather altered pathways governing the macrophage response. In total, we reveal that PTPRD is a bona fide tumor suppressor, pinpoint PTPRD loss as a cause of aberrant STAT3 activation in gliomas, and establish PTPRD loss, in the setting of CDKN2A/p16^INK4A deletion, as a driver of glioma progression.Glioblastoma multiforme (GBM) is a devastating disease. It is the most common and aggressive type of glioma and outcomes remain poor despite current treatments (1). To increase our understanding of the genetic basis of this malignancy, several mutational survey studies examining GBM have been completed and provide a detailed view of the molecular changes underlying this cancer (2–4). Because GBM is a highly heterogeneous tumor, a challenge remains to determine which molecular alterations drive tumorigenesis and to understand the underlying mechanisms of action. Recent work by our group and others have identified inactivation of protein tyrosine phosphatase receptor-δ (PTPRD) as a frequent alteration in GBM and other tumors, and showed that PTPRD copy number loss correlates with poor prognosis (5–10). Despite the high prevalence of PTPRD inactivation in human tumors, it is not known whether loss of PTPRD can promote tumorigenesis. Furthermore, the mechanisms of action and the oncogenic context in which PTPRD acts remain obscure.PTPRD belongs to a family of protein-tyrosine phosphatases that collectively have been implicated in functions, including the regulation of receptor tyrosine kinases, growth, cell migration, and angiogenesis (11). Previously, we demonstrated that phosphorylated STAT3 (p-STAT3) is a substrate of PTPRD and that cancer-specific mutations in PTPRD abrogate the ability of the phosphatase to dephosphorylate STAT3 (5). Interestingly, accumulation of phosphorylated STAT3 and STAT3 hyperactivation are frequent events in solid tumors like GBM, yet the genetic basis of aberrant STAT3 activation is poorly understood. p-STAT3 has been implicated in a number of tumor-promoting processes, including blocking differentiation, maintaining the stem cell pool, promoting growth and angiogenesis, and regulating the immune response and tumor microenvironment (12–14). In this study, we show that allelic loss of Ptprd results in p-Stat3 accumulation and Stat3 hyperactivation, elucidating one genetic root cause for aberrant STAT3 activation in GBM.Chromosome 9p, a region frequently lost in gliomas, contains the genes encoding PTPRD and the cyclin dependent kinase inhibitor 2A (CDKN2A). The CDKN2A locus produces the p16^INK4A and p14/p19^ARF tumor suppressors by alternate splicing (15). We and others have shown that selective pressure exists for inactivation of both PTPRD and CDKN2A, on chromosome 9p, in many types of cancer (5, 6, 10, 16). Both genes are frequently deleted or mutated. In this study, we develop a murine tumor model in which we inactivate both genes to model the genetic events that occur on 9p. We demonstrate that Ptprd loss cooperates with Cdkn2a deletion to promote tumorigenesis.We define the cooperative effects of PTPRD and CDKN2A by using Ptprd knockout and Cdkn2a/p16^Ink4a knockout mice in conjunction with the replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor retrovirus (RCAS) PDGFB/Nestin-tvA glioma mouse model. In this well-established RCAS model, the PDGFB oncogene drives glioma formation. PDGFB is specifically introduced into Nestin-expressing glial progenitor cells via infection of the avian RCAS virus into mice that express the avian tvA receptor under the Nestin promoter (17–19). Intracranial gliomas generated by the RCAS PDGFB/Nestin tvA mouse model reflect the histology of human GBM (20). Furthermore, as opposed to traditional genetically engineered mouse models, genes can be introduced into adult somatic cells of mice with excellent temporal specificity (19). Here, we show that Ptprd is a haploinsufficient tumor suppressor that cooperates with deletion of Cdkn2a/p16^Ink4a to promote glioma progression.     

Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity, which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications, clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however, the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs, most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification, revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well.     

Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer

The underpinnings of STAT3 hyperphosphorylation resulting in enhanced signaling and cancer progression are incompletely understood. Loss-of-function mutations of enzymes that dephosphorylate STAT3, such as receptor protein tyrosine phosphatases, which are encoded by the PTPR gene family, represent a plausible mechanism of STAT3 hyperactivation. We analyzed whole exome sequencing (n = 374) and reverse-phase protein array data (n = 212) from head and neck squamous cell carcinomas (HNSCCs). PTPR mutations are most common and are associated with significantly increased phospho-STAT3 expression in HNSCC tumors. Expression of receptor-like protein tyrosine phosphatase T (PTPRT) mutant proteins induces STAT3 phosphorylation and cell survival, consistent with a “driver” phenotype. Computational modeling reveals functional consequences of PTPRT mutations on phospho-tyrosine–substrate interactions. A high mutation rate (30%) of PTPRs was found in HNSCC and 14 other solid tumors, suggesting that PTPR alterations, in particular PTPRT mutations, may define a subset of patients where STAT3 pathway inhibitors hold particular promise as effective therapeutic agents.Tyrosine phosphorylation regulates a multitude of cellular processes by coordinately activating and inactivating signaling proteins. Aberrations of protein tyrosine phosphorylation and signaling are a hallmark for oncogenic events found in most human cancers. The phosphorylation/dephosphorylation of tyrosine residues on signaling proteins is directly mediated by protein tyrosine kinases and phosphatases. Although many cellular factors are known to dynamically control the activity of these enzymes, genetic alterations of kinases and phosphatases in human cancers lead to perturbations in the levels of tyrosine phosphorylated proteins, uncontrolled cell growth, and tumor formation. Although activating mutations of tyrosine kinases have been extensively studied (1, 2), cancer-associated mutations of tyrosine phosphatases remain incompletely understood, partly due to the lack of comprehensive genomic analysis of these large arrays of phosphatases, as well as their largely unknown and often ambiguous actions in normal physiology and cancer biology.Among the 107 known protein tyrosine phosphatases, the receptor protein tyrosine phosphatases (RPTPs) represent the largest family of the human tyrosine phosphatome, comprising 21 family members (3). These RPTPs are believed to be crucial for the regulation of inter- as well as intracellular signaling due to the cell-surface localization of RPTPs. Selected members of the RPTP family have been reported to function as tumor suppressors, where gene mutation, deletion, or methylation may contribute to the cancer phenotype (3).STAT3 is an oncogene, and constitutive STAT3 activation is a hallmark of human cancers. Activating STAT3 mutations are rare in all cancers studied to date, including head and neck squamous cell carcinoma (HNSCC) (4). Although activating mutations of upstream receptor tyrosine kinases leading to increased STAT3 phosphorylation characterize some malignancies [e.g., EGFR mutations in non-small cell lung cancer (5)], most cancers lack these alterations yet harbor elevated phospho-STAT3 (p-STAT3) levels. We (Z.W.) previously reported that STAT3 serves as a substrate for wild-type PTPRT enzyme (also known as RPTP-T, RPTPρ, or RPTPrho) in colorectal cancer cells (SW480 and HT29) and HEK293T cells (6). STAT3 has additionally been reported to be a substrate of PTPRD (also known as PTPδ) in glioblastoma models, suggesting that several RPTP family members may exhibit tumor-suppressive function by dephosphorylating STAT3 (7). In the present study, we hypothesized that mutation of RPTP family members, including PTPRT, results in elevated expression levels of tyrosine phosphorylated STAT3 in human HNSCC. Analysis of reverse-phase protein array and sequencing data demonstrated significant association between PTPR mutation and increased p-STAT3 expression levels in HNSCC, establishing the de novo signaling consequence of PTPR mutations on a major oncogenic pathway that drives human carcinogenesis. Studies in HNSCC models demonstrate that PTPRT mutations induce p-STAT3 and HNSCC survival, consistent with a “driver” phenotype whereas computational modeling revealed functional implications of PTPR mutations on phospho-tyrosine (p-Tyr)–substrate interactions. Analysis of whole-exome sequencing results of 374 primary head and neck squamous cell carcinomas (HNSCCs) revealed that PTPR genes are mutated in nearly one-third (30.7%) of HNSCC tumors, compared with a 15.2% mutation rate in the cytoplasmic protein tyrosine phosphatase (PTP) family. This pattern is strikingly consistent across an additional 14 types of solid tumors sequenced to date, implicating a potentially important pathologic contribution of PTPR mutations to human carcinogenesis. These cumulative findings suggest that genetic alterations of selected PTPRs, including PTPRT, may induce STAT3 activation and serve as predictive biomarkers for treatment with emerging STAT3 pathway inhibitors.     

Low-level microsatellite instability phenotype in sporadic glioblastoma multiforme

Purpose Genetic instability is a hallmark of glioblastoma multiforme (GBM). Microsatellite instability (MSI) is a significant event in the tumorigenesis of many sporadic malignancies. The aim of our investigation was to study microsatellite instability in newly diagnosed glioblastomas.Methods MSI was investigated in 109 GBMs with 15 microsatellite markers. Immunohistochemistry was performed for the mismatch repair (MMR) proteins hMLH1, hMSH2, hPMS2, and hMSH6 in cases showing MSI. Sequence and promoter methylation status of hMLH1 were analyzed in the case of a decreased hMLH1 protein expression as well. To further investigate MSI(+) GBMs we carried out studies of LOH at selected chromosome regions, EGFR amplification, and sequence of p53 and PTEN.Results MSI was observed in six GBMs (5.5%) and it was more frequent in GBMs with a previous lower grade astrocytoma (18.8% vs. 3.2%). MMR protein staining was positive in all MSI(+) GBMs except in one case, which showed an aberrant expression of hMLH1 and hPMS2 without hMLH1 inactivation. Among MSI(+) GBMs, one tumor corresponded to the GBM molecular type 1 (p53 mutation, no EGFR amplification), another tumor to type 2 (wild-type p53, EGFR amplification), and four tumors to neither type (wild-type p53, no EGFR amplification). None of the six tumors carried a PTEN mutation.Conclusions MSI in GBM might be caused by inactivation of minor MMR genes rather than by a deficiency of hMLH1 or hMSH2 and it appears not to play a decisive role in the pathogenesis of these tumors. MSI(+) GBMs predominantly showed a profile which included wild-type of p53 and PTEN and absence of EGFR amplification but MSI occurred in all GBM molecular subtypes.Grant sponsor: supported by a grant from the Deutsche Forschungsgemeinschaft, DFG (MA 2448/1)     

Pattern of retinoblastoma pathway inactivation dictates response to CDK4/6 inhibition in GBM

Glioblastoma multiforme (GBM) is a fatal primary brain tumor harboring myriad genetic and epigenetic alterations. The recent multidimensional analysis of the GBM genome has provided a more complete view of the landscape of such alterations and their linked pathways. This effort has demonstrated that certain pathways are universally altered, but that the specific genetic events altered within each pathway can vary for each particular patient''s tumor. With this atlas of genetic and epigenetic events, it now becomes feasible to assess how the patterns of mutations in a pathway influence response to drugs that are targeting such pathways. This issue is particularly important for GBM because, in contrast to other tumor types, molecularly targeted therapies have failed to alter overall survival substantially. Here, we combined functional genetic screens and comprehensive genomic analyses to identify CDK6 as a GBM oncogene that is required for proliferation and viability in a subset of GBM cell lines and tumors. Using an available small molecule targeting cyclin-dependent kinases (CDKs) 4 and 6, we sought to determine if the specific pattern of retinoblastoma pathway inactivation dictated the response to CDK4/6 inhibitor therapy. We showed that codeletion of CDKN2A and CDKN2C serves as a strong predictor of sensitivity to a selective inhibitor of CDK4/6. Thus, genome-informed drug sensitivity studies identify a subset of GBMs likely to respond to CDK4/6 inhibition. More generally, these observations demonstrate that the integration of genomic, functional and pharmacologic data can be exploited to inform the development of targeted therapy directed against specific cancer pathways.     

Gene expression profiling reveals molecularly and clinically distinct subtypes of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by genetic instability, intratumoral histopathological variability, and unpredictable clinical behavior. We investigated global gene expression in surgical samples of brain tumors. Gene expression profiling revealed large differences between normal brain samples and tumor tissues and between GBMs and lower-grade oligodendroglial tumors. Extensive differences in gene expression were found among GBMs, particularly in genes involved in angiogenesis, immune cell infiltration, and extracellular matrix remodeling. We found that the gene expression patterns in paired specimens from the same GBM invariably were more closely related to each other than to any other tumor, even when the paired specimens had strikingly divergent histologies. Survival analyses revealed a set of approximately 70 genes more highly expressed in rapidly progressing tumors that stratified GBMs into two groups that differed by >4-fold in median duration of survival. We further investigated one gene from the group, FABP7, and confirmed its association with survival in two unrelated cohorts totaling 105 patients. Expression of FABP7 enhanced the motility of glioma-derived cells in vitro. Our analyses thus identify and validate a prognostic marker of both biologic and clinical significance and provide a series of putative markers for additional evaluation.     

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.     

Overexpression of Protein Phosphatase Non-receptor Type 11 (PTPN11) in Gastric Carcinomas

Background  

Tyrosine phosphorylation and dephosphorylation by protein tyrosine kinases and phosphatases (PTPs), respectively, play crucial roles in cellular signal transduction. Protein phosphatase non-receptor type 11 (PTPN11) is a positive signaling PTP that activates RAS and ERK signaling. Also, the PTPN11 binds with CagA of Helicobacter pylori in gastric epithelial cells.     

Protein tyrosine phosphatases and signalling

A cornerstone of many cell-signalling events rests on reversible phosphorylation of tyrosine residues on proteins. The reversibility relies on the coordinated actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), both of which exist as large protein families. This review focuses on the rapidly evolving field of the PTPs. We now know that rather than simply scavenging phosphotyrosine, the PTPs specifically regulate a wide range of signalling pathways. To illustrate this and to highlight current areas of agreement and contention in the field, this review will present our understanding of PTP action in selected areas and will present current knowledge surrounding the regulatory mechanisms that control PTP enzymes themselves. It will be seen that PTPs control diverse processes such as focal adhesion dynamics, cell-cell adhesion and insulin signalling, and their own actions are in turn regulated by dimerisation, phosphorylation and reversible oxidation.     

Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells

Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. Patients with GBM have a uniformly poor prognosis, with a mean survival of 1 y (1). Thus, advances on all fronts, both basic and applied, are needed to combat this deadly disease better. Recent studies have provided evidence for self-renewing, stem-like cells within human gliomas (2). These glioma-initiating cells constitute a small minority of neoplastic cells within a tumor and are defined operationally by their ability to seed new tumors (3). To target these rare glioma-initiating cells, a better understanding of the molecular mechanisms that regulate their formation is essential.Considerable progress has been made in understanding the mutations responsible for GBM. The Cancer Genome Atlas network has cataloged the recurrent genomic abnormalities in GBM by genome-wide DNA copy number events and sequence-based mutation detection for 601 genes (4). Gene expression-based molecular classification has also defined four subtypes of GBM termed proneural, neural, classical, and mesenchymal (5). Proneural GBM is enriched for the oligodendrocyte gene signature, whereas the classical group is associated with the astrocytic signature. The neural class is enriched for genes differentially expressed by neurons, whereas the mesenchymal class is associated with the cultured astroglial signature (5). Several recurrent mutations, such as PDGFRA, IDH1, EGFR, and NF1, also correlate with these GBM subtypes, providing additional support for their existence. Numerous other, often rare, mutations have also been identified in GBM. Although these datasets are valuable for understanding the molecular pathogenesis of GBM, it is still difficult to distinguish between mutations that contributed to tumor initiation and those acquired later during tumor progression.The cell of origin (COO) of GBM is still controversial. Neural stem cells (NSCs) are good candidates because the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for gliomagenesis. NSCs are also more susceptible to malignant transformation than differentiated cells in the adult brain (6, 7). However, the genetic pathways that can transform NSCs into glioma-initiating cells still remain elusive. Transposon-based mutagenesis provides an unbiased, high-throughput method for identifying genes important for GBM (8). Here, we describe a unique two-step insertional mutagenesis strategy that makes it possible to identify genes and signaling pathways that are able to transform a NSC into a cancer-initiating cell for the mesenchymal subtype of GBM. In this two-step approach, NSCs are first mutagenized in vitro and the mutagenized cells are then transplanted into immunocompromised mice for subsequent tumor development following additional rounds of transposon-based mutagenesis. This makes it possible to discriminate between the genetic changes that occur early in tumor initiation and those required for tumor progression. In addition to identifying several previously undescribed GBM candidate cancer genes, our studies suggest that transposon-induced tumors mimic the evolutionary processes now thought to generate many human cancers, in which tumors have a branched cellular and genetic architecture reminiscent of Darwin’s iconic evolutionary tree.     

Therapeutic stem cells expressing variants of EGFR-specific nanobodies have antitumor effects

The deregulation of the epidermal growth factor receptor (EGFR) has a significant role in the progression of tumors. Despite the development of a number of EGFR-targeting agents that can arrest tumor growth, their success in the clinic is limited in several tumor types, particularly in the highly malignant glioblastoma multiforme (GBM). In this study, we generated and characterized EGFR-specific nanobodies (ENb) and imageable and proapoptotic ENb immunoconjugates released from stem cells (SC) to ultimately develop a unique EGFR-targeted therapy for GBM. We show that ENbs released from SCs specifically localize to tumors, inhibit EGFR signaling resulting in reduced GBM growth and invasiveness in vitro and in vivo in both established and primary GBM cell lines. We also show that ENb primes GBM cells for proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Furthermore, SC-delivered immunoconjugates of ENb and TRAIL target a wide spectrum of GBM cell types with varying degrees of TRAIL resistance and significantly reduce GBM growth and invasion in both established and primary invasive GBM in mice. This study demonstrates the efficacy of SC-based EGFR targeted therapy in GBMs and provides a unique approach with clinical implications.The binding of ligands to the epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, leads to activation of the EGFR tyrosine kinase and subsequent stimulation of signal transduction pathways that are involved in regulating cell proliferation, differentiation, migration, and survival (1). Although present in normal cells, EGFR is overexpressed and mutated in a variety of tumors and has been associated with poor prognosis and decreased survival (2). Over the past two decades, much effort has been directed at developing anticancer agents that can interfere with EGFR activity and arrest tumor growth and, in some cases, cause tumor regression. The most commonly used pharmacologic approaches to inhibit EGFR signaling are small-molecule receptor tyrosine kinase inhibitors (smRTKI), like Gefinitib (Iressa, ZD1839) and Erlotinib (Tarceva, OSI-774), and monoclonal antibodies (mAb), such as Cetuximab (Erbitux, Mab-C225), Panitumumab (ABX-EGF), and Matuzumab ({"type":"entrez-protein","attrs":{"text":"EMD72000","term_id":"451921855","term_text":"EMD72000"}}EMD72000). Whereas smRTKI exert their effects at the intracellular domain of EGFR to prevent tyrosine kinase activity, mAbs stearically block ligand binding to the extracellular domain of the receptor (3, 4). Although the use of Erlotinib and Gefitinib have had moderate success in clinical trials in different tumor types, the use of mAbs has had limited to no success in cancer patients (3).One aggressive tumor type with highly overactive EGFR pathway is glioblastoma multiforme (GBM), where the median survival time remains only ∼1 y (5). Gene amplification of the EGFR and activating mutations in EGFR play a significant role in gliomagenesis and can be found in up to 70% of all GBMs (6). The mute response of anti-EGFR therapies in GBMs compared with other tumor types could be mainly attributed to the presence of the blood–brain barrier (BBB), transporter proteins, and catabolism, which are known to severely limit accumulation of the drugs at the tumor site and reduce their therapeutic efficacy (7). Therefore, there is an urgent need to develop EGFR targeting agents and to use innovative modes of delivery to enhance the efficacy of EGFR-targeting therapies for aggressive tumors like GBMs.Recently, antibody-based anticancer therapies that involve smaller antibody fragments such as Fabs, ScFvs and nanobodies have been emerging (8). Nanobodies are derived from heavy chain-only antibodies found in camelids (e.g., Llama glama) and consist solely of the antigen-specific domain (V_HH) (9). These single-domain antibodies are significantly smaller in size (15 kDa) than scFv (28 kDa) or Fab (55 kDa), thereby potentially providing higher tissue dispersion than their counterparts (8). In addition, nanobodies are significantly more stable than V_H domains and have improved penetration against immune-evasive (cryptic) antigens compared with mAbs (10, 11). Nanobodies specific for EGFR have recently been developed and shown to be able to sterically hinder the binding of EGF to the receptor, thereby inhibiting EGFR signaling (12).We and others have shown that both neural stem cells (NSC) and mesenchymal stem cells (MSC) specifically home to tumors (13, 14) and have used this tropism for on-site delivery of therapeutic proteins in mouse tumor models (15, 16). Recently, we have shown the potential of engineered stem cell based therapeutics in a clinically relevant model of tumor resection and invasiveness (17). In this study, we have engineered different bivalent EGFR targeting nanobodies (ENbs) and their imageable and proapoptotic immunoconjugates for extracellular release from stem cells (SC) and extensively characterized them in vitro. Using tumor models of malignant and primary invasive GBM, we have assessed ENb pharmacokinetics in real time and the therapeutic efficacy of ENbs and its proapoptotic immunoconjugate in vivo.     

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-β receptor tyrosine kinase signaling

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1^−/−) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.     

Expression of cyclooxygenase-2 and epidermal growth factor receptor in primary and recurrent glioblastoma multiforme

Purpose: To investigate the pattern and level of cyclooxygenase-2 (COX-2) expression in a series of high grade primary and recurrent glioblastoma multiforme (GBM) and correlation with time to recurrence and patients’ survival following therapy. The relationship between COX-2 and epidermal growth factor receptor (EGFR) immunoreactivities was evaluated. Materials and methods: Specimens of 14 primary and 14 recurrent GBMs (eight pairs) following surgery and full course radiation therapy were processed for immunostaining on COX-2 and EGFR. Tumor cell positivity was semi-quantitatively scored. COX-2 scores of the primary tumor and recurrence were correlated with the time to radiological tumor progression and patients’ survival. Results: COX-2 positive tumor cells were disseminated throughout the tumor parenchyma. The intensity and pattern of COX-2 expression were heterogeneous, with predominant expression in areas surrounding tumor necrosis. Scoring of COX-2 positivity revealed values between 1 and 80% of the cells. Primary GBMs with COX-2 expression levels between 25% and 70% of the tumor cells showed a shorter time to radiological recurrence than GBMs with <10% COX-2 positive tumor cells (respectively, 219±50 and 382±77 days). No correlation was found between the COX-2 expression in the primary tumor and patients’ survival (r _s=−0.073) following therapy. No correlation was found either between COX-2 and EGFR immunoreactivity. Conclusions: Immunohistochemical expression of COX-2 in GBM showed large variation. Hence, determination of COX-2 expression in tumor specimen for each individual might be relevant for selection of those patients, who could benefit from adjuvant therapy with selective COX-2 inhibitors.     

Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake

AB-type toxins, like other bacterial toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The anthrax toxin, however, times its own uptake, suggesting that toxin binding triggers specific signaling events. Here we show that the anthrax toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the anthrax toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.     

Transdifferentiation of glioblastoma cells into vascular endothelial cells

Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53(+/-) heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP(+) vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.     

Calpain‐dependent cleavage of SHP‐1 and SHP‐2 is involved in the dephosphorylation of Jurkat T cells induced by Entamoeba histolytica

Host cell death induced by Entamoeba histolytica is an important mechanism for both host defence and microbial immune evasion during human amoebiasis. However, the signalling pathways underlying cell death induced by E. histolytica are not fully understood. This study investigated the involvement of the protein tyrosine phosphatases (PTPs) SHP‐1 and SHP‐2 in the dephosphorylation associated with E. histolytica‐induced host cell death. Incubation with E. histolytica resulted in a marked decrease in protein tyrosine phosphorylation levels and degradation of SHP‐1 or SHP‐2 in Jurkat cells. Pre‐treatment of cells with a calpain inhibitor, calpeptin, impeded the amoeba‐induced dephosporylation and cleavage of SHP‐1 or SHP‐2. Additionally, inhibition of PTPs with phenylarsine oxide (PAO) attenuated Entamoeba‐induced dephosphorylation and DNA fragmentation in Jurkat T cells. These results suggest that calpain‐dependent cleavage of SHP‐1 and SHP‐2 may contribute to protein tyrosine dephosphorylation in Jurkat T cell death induced by E. histolytica.