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PDF file - 82K, GBM Patient Data (S1); EGFRvIII and CD133 expression in GBM Neurospheres or Normal Brain (NB) derived Neurospheres (S2); Percentage of co-localization of EGFRvIII+ with Markers of Differentiation and Stem/Progenitor Cells (S3).

PDF file - 3725K, Gating Tree for Fluorescence Activated Cell Sorting (FACS) Analysis (S1); Vessel Staining of EGFRvIII in GBM Xenografts (S2); Characterization and Affinity Analysis of the Recombinant Antibodies (S3); Antibody Dependent Cellular Cytotoxicity Induced by BsAb (S4); Limiting Dilution Analysis of Unsorted Tumor Spheres Before and After BsAb Induced ADCC (S5).

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII+/CD133+ defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII+ cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII− cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII+/CD133+ coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII+/CD133+ population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population. Cancer Res; 74(4); 1238–49. ©2013 AACR.

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EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial–mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/β-catenin pathway, leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. Cancer Res; 72(10); 2657–71. ©2012 AACR.

Background Worldwide, socioeconomic differences in health and use of healthcare resources have been reported, even in countries providing universal healthcare coverage. However, it is unclear how large these socioeconomic differences are for different types of care and to what extent health status plays a role. Therefore, our aim was to examine to what extent healthcare expenditure and utilization differ according to educational level and income, and whether these differences can be explained by health inequalities. Methods Data from 18,936 participants aged 25–79 years of the Dutch Health Interview Survey were linked at the individual level to nationwide claims data that included healthcare expenditure covered in 2017. For healthcare utilization, participants reported use of different types of healthcare in the past 12 months. The association of education/income with healthcare expenditure/utilization was studied separately for different types of healthcare such as GP and hospital care. Subsequently, analyses were adjusted for general health, physical limitations, and mental health. Results For most types of healthcare, participants with lower educational and income levels had higher healthcare expenditure and used more healthcare compared to participants with the highest educational and income levels. Total healthcare expenditure was approximately between 50 and 150 % higher (depending on age group) among people in the lowest educational and income levels. These differences generally disappeared or decreased after including health covariates in the analyses. After adjustment for health, socioeconomic differences in total healthcare expenditure were reduced by 74–91 %. Conclusions In this study among Dutch adults, lower socioeconomic status was associated with increased healthcare expenditure and utilization. These socioeconomic differences largely disappeared after taking into account health status, which implies that, within the universal Dutch healthcare system, resources are being spent where they are most needed. Improving health among lower socioeconomic groups may contribute to decreasing health inequalities and healthcare spending.

Dietary supplements are products that are widely used for instance as energisers or to lose weight. There have been cases reported where undeclared ingredients present in such supplements have caused adverse effects on the health of the user. As there are many different products to choose from, it seems impossible to predict which might contain harmful components and to ban them from the market. Nonetheless, the use of dietary supplements and the experiences of users are shared in online discussions. We describe the development of a search engine to retrieve products associated with certain effects. Upon application we were able to retrieve a list of dietary supplements that are repeatedly associated with excessive effects by users on public fora. The top of the list contains supplements that have previously been banned because they contained undeclared harmful components. The use of the search engine as described here is a powerful method for making a risk-based selection of dietary supplements which can then be analysed for the presence of illegal or other unwanted components.

BACKGROUND Nearly half of people who die by suicide see a health care provider in the month before their death. With the release of new care guidelines, detection of suicidal patients will likely increase. Providers need access to suicide-specific resources that can be used as part of immediate, brief interventions with a suicidal patient. Web-based suicide prevention resources have the potential to address this need. OBJECTIVE This study aimed to describe the development of the NowMattersNow.org website as a resource for individuals with suicidal thoughts and to evaluate the utility of the site via user experience surveys. METHODS NowMattersNow.org is an online video-based free public resource that provides evidence-based teachings, examples, and resources for managing suicidal thoughts and intense emotions focused largely around skills from dialectical behavior therapy. Developed with assistance from mental health consumers, it is intended to address gaps in access to services for suicidal patients in health care systems. Visitors stay an average of a minute and a half on the website. From March 2015 to December 2017, a user experience survey measured self-reported changes on a 1 (not at all) to 5 (completely overwhelming) scale regarding intensity of suicidal thoughts and negative emotions while on the website. Longitudinal regression analyses using generalized estimating equations evaluated the magnitude and statistical significance of user-reported changes in suicidal ideation and negative emotion. In secondary analyses, user-reported changes specific to subgroups, including men aged 36 to 64 years, mental health care providers, and other health care providers were evaluated. RESULTS During the period of analysis, there were 138,386 unique website visitors. We analyzed surveys (N=3670) collected during that time. Subsamples included men aged 36 to 64 years (n=512), mental health providers (n=460), and other health care providers (n=308). A total of 28% (1028/3670) of survey completers rated their suicidal thoughts as a 5 or “completely overwhelming” when they entered the website. We observed significant reductions in self-reported intensity of suicidal thoughts (–0.21, P CONCLUSIONS Survey respondents reported measurable reductions in intensity of suicidal thoughts and emotions, including those rating their suicidal thoughts as completely or almost completely overwhelming and among middle-aged men. Although results from this user-experience survey administered at one point in time to a convenience sample of users must be interpreted with caution, results provide preliminary support for the potential effectiveness of the NowMattersNow.org website as a tool for short-term management of suicidal thoughts and negative emotions.

Background: Nearly half of people who die by suicide see a health care provider in the month before their death. With the release of new care guidelines, detection of suicidal patients will likely increase. Providers need access to suicide-specific resources that can be used as part of immediate, brief interventions with a suicidal patient. Web-based suicide prevention resources have the potential to address this need. Objective: This study aimed to describe the development of the NowMattersNow.org website as a resource for individuals with suicidal thoughts and to evaluate the utility of the site via user experience surveys. Methods: NowMattersNow.org is an online video-based free public resource that provides evidence-based teachings, examples, and resources for managing suicidal thoughts and intense emotions focused largely around skills from dialectical behavior therapy. Developed with assistance from mental health consumers, it is intended to address gaps in access to services for suicidal patients in health care systems. Visitors stay an average of a minute and a half on the website. From March 2015 to December 2017, a user experience survey measured self-reported changes on a 1 (not at all) to 5 (completely overwhelming) scale regarding intensity of suicidal thoughts and negative emotions while on the website. Longitudinal regression analyses using generalized estimating equations evaluated the magnitude and statistical significance of user-reported changes in suicidal ideation and negative emotion. In secondary analyses, user-reported changes specific to subgroups, including men aged 36 to 64 years, mental health care providers, and other health care providers were evaluated. Results: During the period of analysis, there were 138,386 unique website visitors. We analyzed surveys (N=3670) collected during that time. Subsamples included men aged 36 to 64 years (n=512), mental health providers (n=460), and other health care providers (n=308). A total of 28% (1028/3670) of survey completers rated their suicidal thoughts as a 5 or “completely overwhelming” when they entered the website. We observed significant reductions in self-reported intensity of suicidal thoughts (–0.21, P

Adoptive immunotherapy using chimeric antigen receptors-modified T cells (CAR-T) is a promising approach for cancer treatment. However, CARs currently applied in the clinics cannot be effectively regulated and the safety of CAR-T cell therapies remains a major concern. To improve the safety of CAR-T cells, we designed a synthetic splitting CAR (ssCAR) that can regulate T cell functions exogenously. Epidermal growth factor receptor variant III (EGFRvIII) was used as a molecular target for ssCAR. Our results indicate that both EGFRvIII and small molecule are needed for the activation of the ssCAR-T cells. AP21967 dose-dependently increased the expression of T cell activation, production of cytokines and extent of cell lysis. In conclusion, the gene switch designed in this study allows for temporal and spatial control over engineered T cells in a dose-and time-dependent manner by AP21967. Our work demonstrates the feasibility and improved safety profile of this novel treatment approach.

BackgroundLarge-scale regeneration programmes to improve the personal conditions and living circumstances in deprived areas may affect health and the lifestyle of the residents. Previous evaluations concluded that a large-scale urban regeneration programme in the Netherlands had some positive effects within 3.5 years. The aim of the current study was to evaluate the effects at the longer run.MethodsWith a quasi-experimental research design we assessed changes in the prevalence of general health, mental health, physical activity, overweight, obesity, and smoking between the pre-intervention (2003–04 –mid 2008) and intervention period (mid 2008–2013–14) in 40 deprived target districts and comparably deprived control districts. We used the Difference-in-Difference (DiD) to assess programme impact. Additionally, we stratified analyses by sex and by the intensity of the regeneration programme.ResultsChanges in health and health related behaviours from pre-intervention to the intervention period were about equally large in the target districts as in control districts. DiD impact estimates were inconsistent and not statistically significant. Sex differences in DiD estimates were not consistent or significant. Furthermore, DiD impact estimates were not consistently larger in target districts with more intensive intervention programmes.ConclusionWe found no evidence that this Dutch urban regeneration programme had an impact in the longer run on self-reported health and related behaviour at the area level.

// Yan Zheng 1, * , Xiu-Ling Li 1, * , Xiang-Dong Sun 1 , Bing-Yong Zhang 1 , Puja Gupta 2 , Albert J Wong 2 , Tian-Fang Li 3 and Shuang-Yin Han 1 1 Translational Research Center, People’s Hospital of Henan Province, Zhengzhou University, Zhengzhou 450003, China 2 Brain Tumor Research Laboratories, Department of Neurosurgery, Stanford University Medical Center, Stanford, CA 94305, USA 3 The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450003, P.R. China * These authors contribute equally to this work Correspondence to: Shuang-Yin Han, email: hansy001@henu.edu.cn Tian-Fang Li, email: tfli@zzu.edu.cn Keywords: chimeric antigen receptor; regulation; glioblastoma Received: July 14, 2017 Accepted: October 30, 2017 Published: January 02, 2018 ABSTRACT Adoptive immunotherapy using chimeric antigen receptors-modified T cells (CAR-T) is a promising approach for cancer treatment. However, CARs currently applied in the clinics cannot be effectively regulated and the safety of CAR-T cell therapies remains a major concern. To improve the safety of CAR-T cells, we designed a synthetic splitting CAR (ssCAR) that can regulate T cell functions exogenously. Here, ssCAR was constructed by splitting recognition and key signaling module into distinct parts, mimicking the natural dual signal input of T cell activation. These separated parts form heterodimer upon stimulation by a small molecule with subsequent activation of ssCAR-T cells. Epidermal growth factor receptor variant III (EGFRvIII), a tumor specific antigen highly expressed in glioblastoma and other human malignancies, was used as a molecular target for ssCAR. Our results indicate that both EGFRvIII and small molecule are needed for the activation of the ssCAR-T cells. AP21967 dose-dependently increased the expression of T cell activation marker CD69, production of cytokines IFN-γ and TNF-α, and extent of cell lysis. When co-cultured with EGFRvIII + U87 in the presence of 500 nM AP21967, ssCAR-T cells showed a significant increase in cytokine production and targeted cells killing capacity to a level comparable to conventional CAR. Increasing the duration of treatment induced a simultaneous up-regulation of apoptotic rate of tumor cells. In conclusion, the gene switch designed in this study allows for temporal and spatial control over engineered T cells in a dose-and time-dependent manner by AP21967. Our work demonstrates the feasibility and improved safety profile of this novel treatment approach.

Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the EGF receptor in many human tumors. This variant is tumor specific and highly immunogenic, thus, it can be used as a target for targeted drug delivery toward tumor cells. The major aim of this study was to develop an EGFRvIII-mediated drug delivery system by anti-EGFRvIII monoclonal antibody (MAb) conjugated to doxorubicin (Dox)-loaded nanostructured lipid carriers (NLC) to enhance the targeting specificity and cytotoxic effect of Dox on EGFRvIII-overexpressing cell line. In our study, Dox was chosen as a hydrophobic cytotoxic drug and drug-loaded nanostructured lipid carriers (Dox-NLC) was prepared by solvent emulsification/evaporation method. In order to conjugate anti-EGFRvIII MAb to Dox-NLC, DSPE-PEG2000-NHS (1,2-distearoylphosphatidylethanolamine-polyethylene glycol 2000-NHS) was used as a linker. Physicochemical characteristics of antibody conjugated Dox-NLC (MAb-Dox-NLC), including particle size, zeta potential, entrapment efficiency and in vitro Dox release were investigated. Cytotoxicity of MAb-Dox-NLC against NIH-3T3 and HC2 20d2/c (EGFRvIII-transfected NIH-3T3) cell lines was evaluated. The MAb-Dox-NLC appeared to enhance the cytotoxic activity of targeted NLC against HC2 20d2/c cells. The cellular uptake percentage of targeted NLC by HC2 20d2/c cells was higher than that of NIH-3T3 cells, indicating that EGFRvIII can specifically target HC2 20d2/c cells. In conclusion, anti-EGFRvIII MAb-targeted NLC may be considered as an effective nanocarrier for targeted drug delivery.

A large proportion of GBM tumors express an altered, constitutively active version of the EGF receptor referred to as EGFRvIII. Along with increasing proliferation and inhibiting apoptosis, expression of EGFRvIII is also a GBM stem cell marker. Targeting tumor stem cells based on EGFRvIII expression may therefore improve survival for GBM patients. As such, several clinical trials using a peptide vaccine directed against EGFRvIII have shown promising results. We have explored improved versions of our original EGFRvIII peptide vaccine and have identified a candidate that shows a 60% improvement in murine subcutaneous tumor models. In this study, we further assessed the efficacy of this 2nd generation vaccine in a murine GL261 intracranial tumor model. Mice that received the EGFRvIII peptide vaccine survived 40% longer (median) than non-vaccinated mice. This response required both CD4+ and CD8+ T cells as the observed survival benefit was lost when either of these cell populations was depleted. The specificity of this vaccine strategy was valicated by an in vitro cytotoxicity assay which demonstrated that T cells derived from EGFRvIII peptide vaccinated mice were capable of specifically killing EGFRvIII expressing GL261 target cells in vitro, while demonstrating no cytotoxitiy against EGFRvIII negative cells. Treatment with the peptide vaccine increased the abundance of tumor infiltrating CD8+ T cells (p=0.0017) and altered the intra-tumoral CD4+ to CD8+ cell ratio (p=0.0015). Moreover, relative to control mice, tumor infiltrating CD8+ T cells in vaccinated mice show significant expression of the conventional immune checkpoint receptor PD-1, indicating that combinational therapies may potentiate the vaccine induced survival benefit in this model. We are currently assessing the utility of both adjuvant and immunomodulatory combinations in an attempt to further increase the efficacy of this second generation peptide vaccine.

Background:The human epidermal growth factor receptor (EGFR) is an important therapeutic target in oncology, and three different types of EGFR inhibitors have been approved for the treatment of cancer patients. However, there has been no clear association between the expression levels of EGFR protein in the tumours determined by the FDA-approved EGFR PharmDx kit (Dako) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors.Method:In this study, we investigated the potential of our anti-EGFR monoclonal antibodies (mAbs; ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild-type EGFR and/or the type-III deletion mutant form of EGFR (EGFRvIII) in formalin-fixed, paraffin-embedded human tumour specimens.Results:We found that the anti-EGFR mAb in the EGFR PharmDx kit stained both wild-type and EGFRvIII-expressing cells in formalin-fixed, paraffin-embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR10 and ICR9 were specific for the wild-type EGFR.Conclusion:We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild-type EGFR protein in tumour specimens using immunohistochemistry, and to determine their prognostic significance, as well as predictive value for response to therapy with EGFR antibodies.British Journal of Cancer advance online publication, 7 February 2012; doi:10.1038/bjc.2012.27 www.bjcancer.com.

Glioblastoma multiforme (GBM) is the most common and deadly of the human brain cancers. The EGF receptor is often amplified in GBM and provides a potential therapeutic target. However, targeting the normal receptor is complicated by its nearly ubiquitous and high level of expression in certain tissues. A naturally occurring deletion mutant of the EGF receptor, EGFRvIII, is a constitutively active variant originally identified in a high percentage of brain cancer cases, and more importantly is rarely found in normal tissue. A peptide vaccine, rindopepimut (CDX-110, Celldex Therapeutics), is directed against the novel exon 1-8 junction produced by the EGFRvIII deletion, and it has shown high efficacy in preclinical models. Recent Phase II clinical trials in patients with newly diagnosed GBM have shown EGFRvIII-specific immune responses and significantly increased time to progression and overall survival in those receiving vaccine therapy, as compared with published results for standard of care. Rindopepimut therefore represents a very promising therapy for patients with GBM.

An anti-EGFRvIII peptide vaccine for treating glioblastoma (Rindopepimut) has shown significant clinical potential, recently receiving Breakthrough Therapy Designation from the FDA. Interestingly, we created this vaccine before MHC binding principles were understood so the sequence was arbitrarily chosen and never optimized for immune response. X-ray diffraction of the EGFRvIII peptide reveals that it has structure, unusual for a 13 amino acid peptide, and the novel glycine at the junction, thought to be essential, is not critical for immune recognition. However, the glycine is at a hinge so we hypothesized this position is important for secondary structure. In our EGFRvIII/NIH-3T3 syngeneic tumor model, we tested substitution of the glycine by all 19 other amino acids, length variations, synthetic amino acids and deletion of the glycine (-Gly) (N = 16 or greater for 24 peptides tested). Several substitutions, including Ala, Val, Pro, show a >58% improved survival rate over Rindopepimut. Most substitutions were similar to Rindopepimut, but some, including His, Cys and -Gly, were comparable to control. All peptides elicited antibodies specific for EGFRvIII with a general trend towards high titer (>1:2x105) in the higher performing vaccines. CTL responses by Elispot revealed that the highest performing vaccines elicited a >42% increase in spots over Rindopepimut (p 19 cleavage products, including recombination to form a larger products at ∼2600Da. Rindopepimut produced ∼14 fragments with lower amounts of the 2600Da, while the poorest vaccines showed almost no cleavage and no 2600Da. Our results show that more robust vaccines against EGFRvIII can be produced, that 3D structure is important, and the vaccine shows unexpected complex processing. Further work is being performed to bring these vaccines to clinical trial.

The epidermal growth factor receptor (EGFR) is essential to multiple physiological and neoplastic processes via signaling by its tyrosine kinase domain and subsequent activation of transcription factors. EGFR overexpression and alteration, including point mutations and structural variants, contribute to oncogenesis in many tumor types. In this study, we identified an in-frame splice variant of the EGFR called mini-LEEK (mLEEK) that is more broadly expressed than the EGFR and is overexpressed in several cancers. Unlike previously characterized EGFR variants, mLEEK lacks the extracytoplasmic, transmembrane and tyrosine kinase domains. mLEEK localizes in the nucleus and functions as a transcription factor to regulate target genes involved in the cellular response to endoplasmic reticulum (ER) stress, including the master regulator of the unfolded protein response (UPR) pathways, molecular chaperone GRP78/Bip. We demonstrated that mLEEK regulates GRP78 transcription through direct interaction with a cis-regulatory element within the gene promoter. Several UPR pathways were interrogated and mLEEK expression was found to attenuate the induction of all pathways upon ER stress. Conversely, knockdown of mLEEK resulted in caspase-mediated cell death and sensitization to ER stress. These findings indicate that mLEEK levels determine cellular responses to unfavorable conditions that cause ER stress. This information, along with the overexpression of mLEEK in tumors, suggests unique strategies for therapeutic intervention. Furthermore, the identification of mLEEK expands the known mechanisms by which the EGFR gene contributes to oncogenesis and represents the first link between two previously disparate areas in cancer cell biology: EGFR signaling and the UPR.

The authors describe 3 studies in which hypnosis itself is not studied but instead used to create anomalous states in the laboratory that can be studied under controlled conditions. The 1st article is a comprehensive review of programmatic research using hypnosis to elicit and study clinically relevant delusions. The 2nd article reviews studies comparing the brain activity of hysterical/dissociative patients with nonpatients hypnotized and given suggestions for sensory-motor and cognitive anomalies typical of the clinical syndromes. The authors conclude that the hypnosis analogues are relevant and revealing. The 3rd article describes a single experiment using hypnosis to elicit distressing and intrusive memories, typical of acute anxiety disorders. Findings with hypnotic subjects are in keeping with those from patients suffering intrusive memories. Across all 3 papers, hypnosis is shown to be a viable and helpful tool for experimental psychopathology.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.

Activation of the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator.

c-Jun NH2-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and have been implicated in the formation of several human tumors, especially gliomas. We have previously shown that a 55 kDa JNK isoform is constitutively active in 86% of human brain tumors and then showed that it is specifically a JNK2 isoform and likely to be either JNK2α2 or JNK2β2. Notably, we found that only JNK2 isoforms possess intrinsic autophosphorylation activity and that JNK2α2 has the strongest activity. In the present study, we have further explored the contribution of JNK2 isoforms to brain tumor formation. Analysis of mRNA expression by reverse transcription-PCR revealed that JNK2α2 is expressed in 91% (10 of 11) of glioblastoma tumors, whereas JNK2β2 is found in only 27% (3 of 11) of tumors. Both JNK2α2 and JNK2β2 mRNAs are expressed in normal brain (3 of 3). Using an antibody specific for JNK2α isoforms, we verified that JNK2α2 protein is expressed in 88.2% (15 of 17) of glioblastomas, but, interestingly, no JNK2α2 protein was found in six normal brain samples. To evaluate biological function, we transfected U87MG cells with green fluorescent protein–tagged versions of JNK1α1, JNK2α2, and JNK2α2APF (a dominant-negative mutant), and derived cell lines with stable expression. Each cell line was evaluated for various tumorigenic variables including cellular growth, soft agar colony formation, and tumor formation in athymic nude mice. In each assay, JNK2α2 was found to be the most effective in promoting that phenotype. To identify effectors specifically affected by JNK2α2, we analyzed gene expression. Gene profiling showed several genes whose expression was specifically up-regulated by JNK2α2 but down-regulated by JNK2α2APF, among which eukaryotic translation initiation factor 4E (eIF4E) shows the greatest change. Because AKT acts on eIF4E, we also examined AKT activation. Unexpectedly, we found that JNK2α2 could specifically activate AKT. Our data provides evidence that JNK2α2 is the major active JNK isoform and is involved in the promotion of proliferation and growth of human glioblastoma tumors through specific activation of AKT and overexpression of eIF4E. (Cancer Res 2006; 66(20): 10024-31)

The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The β1C and β1A integrins, two cytoplasmic variants of the β1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the β1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, β1A does not affect IGF-II levels. We provide evidence that β1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the β1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the β1 cytoplasmic domain via activation of the growth factor receptor binding protein 2–associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of β1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of β1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype. (Cancer Res 2006; 66(1): 331-42)

Summary The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.

Upon the addition of different growth factors and cytokines, the Gab1 docking protein is tyrosine phosphorylated and in turn activates different signaling pathways. On the basis of the large body of evidence concerning cross talk between the signaling pathways activated by growth factors and oxidative stress, we decided to investigate the role of Gab1 in oxidative injury. We stimulated wild-type mouse embryo fibroblasts (MEF) or MEF with a homozygous deletion of the Gab1 gene (-/- MEF) with H(2)O(2). Our results show that Gab1 is phosphorylated in a dose- and time-dependent manner after H(2)O(2) triggering. Gab1 then recruits molecules such as SHP2, phosphatidylinositol 3-kinase (PI3K), and Shc. Gab1 phosphorylation is sensitive to the Src family kinase inhibitor PP2. Furthermore, we demonstrate that Gab1 is required for H(2)O(2)-induced c-Jun N-terminal kinase (JNK) activation but not for ERK2 or p38 activation. Reconstitution of Gab1 in -/- MEF rescues JNK activation, and we find that this is dependent on the SHP2 binding site in Gab1. Cell viability assays reveal that Gab1 has a dual role in cell survival: a positive one through its interaction with PI3K and a negative one through its interaction with SHP2. This is the first report identifying Gab1 as a component in oxidative stress signaling and one that is required for JNK activation.

With a view to their use in cancer therapy, we have produced rat monoclonal antibodies (MAbs) directed against 5 distinct epitopes (A-E) on the external domain of the wild-type human EGF receptor (EGFR). Here, we have investigated the relative binding and anti-tumour activity of our anti-EGFR MAbs against HC2 20d2/c cells, which have been engineered to overexpress the type-III mutated form of the human EGFR (EGFRvIII). We found that anti-EGFR MAbs that are the most effective antagonists of EGFR ligands (e.g., ICR16, ICR62 and ICR80) also bind to cells that overexpress the EGFRvIII. Although these antibodies are potent inhibitors of the growth of cells which express wild-type EGFR, they did not directly inhibit the growth in vitro of EGFRvIII expressing HC2 20d2/c cells, or the constitutive tyrosine kinase activity of this receptor. However, in the presence of human peripheral blood mononuclear cells (PBMC), the rat IgG2b MAb ICR62 induced strong antibody-dependent cell-mediated cytotoxicity (ADCC) against HC2 20d2/c cells in culture. Interestingly, MAb ICR62 also inhibited very effectively experimental lung metastases of HC2 20d2/c cells in athymic nude mice. Our results suggest that anti-EGFR MAb ICR62, which binds to the EGFRvIII, may have potential in the treatment of tumors which overexpress the EGFRvIII via immunological mechanisms such as ADCC. Since tumours that are EGFRvIII positive may also overexpress the wild-type EGFR, the use of anti-EGFR MAbs that target both wild-type and mutant receptors may have advantages over those that target only1form.

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate–responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

Storage of unstained paraffin slides may lead to the deterioration of specimens and failure to detect cellular proteins immunohistochemically. Although the implication of age-induced alterations on multicenter immunohistochemical studies would be considerable, they have not been investigated previously. The current study was undertaken to examine the effect of this factor further and to explore new ways of overcoming the resultant shortcomings. The authors now report on the immunodetection of a host of antigens in similarly preserved unstained serial paraffin slides obtained from three centers using a panel of eight antibodies. Staining of recently prepared sections from the authors' centers resulted in similar strong patterns in seven of eight antibodies, with one antibody demonstrating variable immunoreactivity. However, storage of unstained paraffin sections at room temperature resulted in a variable but progressive decrease in expression of several tissue antigens. Although the loss in antigenicity was proportional to the length of storage, the effect was reversible if super antibody concentrations were used. The authors conclude that recently prepared paraffin sections from centers with similar fixation protocols have similar immunoreactivity and are suitable for use in comparative multicenter studies. However, in view of the delays that may attend tissue transportation during these projects, the authors suggest that test systems should be checked for age-induced antigen degradation by incubating sections with higher antibody concentrations.

The Grb2-associated binder-1 (Gab1) is one of the major adapter molecules downstream of growth factor receptor signaling. Even though insulin causes tyrosine phosphorylation of Gab1, its role in insulin signaling has not been identified yet. We have demonstrated that insulin increased expression of early growth response gene-1 (egr-1), which is one of the most important transcription factors involved in cell proliferation and differentiation. In the present study, the possible role of Gab1 in insulin-induced egr-1 expression was studied using Rat1 fibroblasts expressing human insulin receptors and wildtype Gab1 (HIRc/Gab1(WT)), Gab1 with three tyrosines in the phosphatidylinositol (PI) 3'-kinase binding domain mutated to phenylalanine (HIRc/Gab1(DeltaPI3K)), or histidinol resistance only (HIRc/HIS). Insulin-induced egr-1 expression in HIRc/Gab1(DeltaPI3K) cells was much lower than in the other cells, as determined by Northern blot analysis. These results suggest that Gab1 is involved in the signaling pathway for insulin-induced egr-1 expression through increasing PI3'-kinase activity. The MAP kinase activity increased less with insulin treatment in HIRc/Gab1(DeltaPI3K) cells than in other cells. Inhibition of MAP kinase by the MEK inhibitor completely abolished insulin-induced egr-1 expression. These results suggest that Gab1 increases MAP kinase activity through its PI3'-kinase binding site, which then leads to egr-1 expression. Our results indicate that Gab1 is involved in the control of egr-1 expression regulated by insulin.

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII+/CD133+ defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII+ cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII− cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII+/CD133+ coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII+/CD133+ population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population. Cancer Res; 74(4); 1238–49. ©2013 AACR.

Earlier studies have demonstrated an unexplained depletion of the epidermal growth factor receptor (EGFR) protein expression in prostatic cancer. We now attribute this phenomenon to the presence of a variant EGFR (EGFRvIII) that is highly expressed in malignant prostatic neoplasms. In a retrospective study, normal, benign hyperplastic and malignant prostatic tissues were examined at the mRNA and protein levels for the presence of this mutant receptor. The results demonstrated that whilst EGFRvIII was not present in normal prostatic glands, the level of expression of this variant protein increased progressively with the gradual transformation of the tissues to the malignant phenotype. The selective association of high EGFRvIII levels with the cancer phenotype underlines the role that this mutant receptor may maintain in the initiation and progression of malignant prostatic growth, and opens the way for new approaches in the management of this disease including gene therapy. © 2000 Cancer Research Campaign

The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.

Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.

Hepatocyte growth factor/scatter factor is a multifunctional factor that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Met-tyrosine kinase. Upon stimulation with hepatocyte growth factor, the Met beta subunit becomes highly phosphorylated on tyrosine residues, one of which, tyrosine 1356 within the carboxyl terminus, is crucial for dissociation, motility, and branching tubule formation in Madin-Darby canine kidney epithelial cells. Tyrosine 1356 forms a multisubstrate binding site for the Grb2 and Shc adaptor proteins, the p85 subunit of phosphatidylinositol 3'-kinase, phospholipase Cgamma, and a phosphatase, SHP2. To investigate additional signaling molecules that are activated by the Met receptor, we have identified hepatocyte growth factor-induced phosphoproteins in tubular epithelial cells. We have established that proteins of 100-130 kDa are highly phosphorylated following stimulation of epithelial cells and that one of these is the Grb2-associated binding protein Gab1, a possible insulin receptor substrate-1-like signal transducer. We show that Gab1 is the major substrate for the Met kinase in vitro and in vivo. Association of Gab1 with Met requires a functional Grb2 binding site involving tyrosine 1356 and to a lesser extent tyrosine 1349. Met receptor mutants that fail to induce branching tubulogenesis are impaired in their ability to interact with Gab1, suggesting that Gab1 may play a role in these processes.

Mutation of the autophosphorylation sites of receptor protein-tyrosine kinases alters ligand dependent internalization and down-regulation, indicating a critical role for these sites in receptor processing. Currently, no differences in receptor processing based on an individual autophosphorylation site have been defined. By using a glutathione S-transferase fusion protein containing the src homology 2 domains of phospholipase C-gamma1 to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P)992), we have found differences in this subpopulation of receptors. Following EGF stimulation, the number of Tyr(P)992 receptors increased 2-fold over receptors identified by an antibody that recognizes activated EGF receptors (alpha-Act. EGFR) in A431 cells. Confocal fluorescence microscopy showed that Tyr(P)992 receptors underwent endocytosis at a slower rate and did not rapidly concentrate in juxtanuclear bodies. Tyr(P)992 receptors were associated with more SOS, Ras-GTPase activating protein, phosphatidylinositol 3-kinase, and SHPTP2/syp, but less Grb2, than receptors in the general population, and these receptors were more heavily phosphorylated than the general population of active receptors. These findings suggest that autophosphorylation status is relevant to the endocytosis, degradation, and effector molecule interaction of individual EGF receptors. Further investigations based on phosphorylation status should provide new insights into how receptor protein-tyrosine kinase signaling is regulated.

Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis., Author Summary Gene fusions represent an important class of cancer genes, created by rearrangements of the genome that bring together two different genes. Because they are unique to cancer cells, gene fusions are ideal diagnostic markers and therapeutic targets. While gene fusions were once thought restricted mainly to blood cancers, recent discoveries suggest they are more widespread. Here, we have developed an approach for mining DNA microarray data to detect the tell-tale signatures of gene fusions, as “breakpoints” occurring within the encoding DNA or expressed transcripts. We apply this approach to a large collection of nearly 1,000 human cancer specimens. From this analysis, we discover and verify twelve new gene fusions occurring in diverse cancer types. We verify that some of these rearrangements recur in other samples of the same cancer type (supporting a causal role) and that the cancers show dependency on the fusion for cancer cell growth. Notably, some of these fusions (e.g. CEP85L/ROS1 in angiosarcoma) represent the first for that cancer type and thus provide important new biological insight. Some are also good drug targets (including rearrangements of ROS1, RAF1, and CDK6 kinases), with clear implications for therapy.

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. Conclusions Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFRvIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D. Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong, and A. J. Wong, submitted for publication). In this report, we document that expression of EGFRvIII in fibroblasts is associated with downstream activation of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (MEK) and modest activation of p42 and p44 MAP kinases. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol 12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFRvIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. Our results suggest that EGFRvIII constitutively activates downstream signal transduction through MAP kinase, and this chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal.

Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.

EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial–mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/β-catenin pathway, leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. Cancer Res; 72(10); 2657–71. ©2012 AACR.

Despite numerous clinical trials over the past 2 decades, the overall survival for children diagnosed with diffuse intrinsic pontine glioma (DIPG) remains 9–10 months. Radiation therapy is the only treatment with proven effect and novel therapies are needed. Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the epidermal growth factor receptor and is expressed in many tumor types but is rarely found in normal tissue. A peptide vaccine targeting EGFRvIII is currently undergoing investigation in phase 3 clinical trials for the treatment of newly diagnosed glioblastoma (GBM), the tumor in which this variant receptor was first discovered. In this study, we evaluated EGFRvIII expression in pediatric DIPG samples using immunohistochemistry with a double affinity purified antibody raised against the EGFRvIII peptide. Staining of pediatric DIPG histological samples revealed expression in 4 of 9 cases and the pattern of staining was consistent with what has been seen in EGFRvIII transfected cells as well as GBMs from adult trials. In addition, analysis of tumor samples collected immediately post mortem and of DIPG cells in culture by RT-PCR, western blot analysis, and flow cytometry confirmed EGFRvIII expression. We were therefore able to detect EGFRvIII expression in 6 of 11 DIPG cases. These data suggest that EGFRvIII warrants investigation as a target for these deadly pediatric tumors.