Albert J. Wong, M.D.

All Publications

• Targeting a Glioblastoma Cancer Stem-Cell Population Defined by EGF Receptor Variant III. Cancer research Emlet, D. R., Gupta, P., Holgado-Madruga, M., Del Vecchio, C. A., Mitra, S. S., Han, S., Li, G., Jensen, K. C., Vogel, H., Xu, L. W., Skirboll, S. S., Wong, A. J. 2014; 74 (4): 1238-1249

  Abstract

  The relationship between mutated proteins and the cancer stem cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an 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 co-expressed with CD133 and that EGFRvIII+/CD133+ defines the population of cancer stem cells with the highest degree of self-renewal and tumor initiating ability. EGFRvIII+ cells are associated with other stem/progenitor markers while 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+ co-expression and 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.

  View details for DOI 10.1158/0008-5472.CAN-13-1407

  View details for PubMedID 24366881

• EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms. Oncogene Del Vecchio, C. A., Giacomini, C. P., Vogel, H., Jensen, K. C., Florio, T., Merlo, A., Pollack, J. R., Wong, A. J. 2013; 32 (21): 2670-2681

  Abstract

  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.

  View details for DOI 10.1038/onc.2012.280

  View details for PubMedID 22797070

• Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma JOURNAL OF HEMATOLOGY & ONCOLOGY Shen, C., Yang, Y., Han, E. Q., Cao, N., Wang, Y., Wang, Y., Zhao, Y., Zhao, L., Cui, J., Gupta, P., Wong, A. J., Han, S. 2013; 6

  Abstract

  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.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.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.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.

  View details for DOI 10.1186/1756-8722-6-33

  View details for Web of Science ID 000319315600001

  View details for PubMedID 23656794

  View details for PubMedCentralID PMC3658918

• Breakpoint analysis of transcriptional and genomic profiles uncovers novel gene fusions spanning multiple human cancer types. PLoS genetics Giacomini, C. P., Sun, S., Varma, S., Shain, A. H., Giacomini, M. M., Balagtas, J., Sweeney, R. T., Lai, E., Del Vecchio, C. A., Forster, A. D., Clarke, N., Montgomery, K. D., Zhu, S., Wong, A. J., van de Rijn, M., West, R. B., Pollack, J. R. 2013; 9 (4)

  Abstract

  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.

  View details for DOI 10.1371/journal.pgen.1003464

  View details for PubMedID 23637631

  View details for PubMedCentralID PMC3636093

• Breakpoint analysis of transcriptional and genomic profiles uncovers novel gene fusions spanning multiple human cancer types. PLoS genetics Giacomini, C. P., Sun, S., Varma, S., Shain, A. H., Giacomini, M. M., Balagtas, J., Sweeney, R. T., Lai, E., Del Vecchio, C. A., Forster, A. D., Clarke, N., Montgomery, K. D., Zhu, S., Wong, A. J., van de Rijn, M., West, R. B., Pollack, J. R. 2013; 9 (4)

  View details for DOI 10.1371/journal.pgen.1003464

  View details for PubMedID 23637631

• Expression of epidermal growth factor variant III (EGFRvIII) in pediatric diffuse intrinsic pontine gliomas JOURNAL OF NEURO-ONCOLOGY Li, G., Mitra, S. S., Monje, M., Henrich, K. N., Bangs, C. D., Nitta, R. T., Wong, A. J. 2012; 108 (3): 395-402

  Abstract

  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.

  View details for DOI 10.1007/s11060-012-0842-3

  View details for Web of Science ID 000305123800007

  View details for PubMedID 22382786

• A novel epidermal growth factor receptor variant lacking multiple domains directly activates transcription and is overexpressed in tumors ONCOGENE Piccione, E. C., LIEU, T. J., Gentile, C. F., Williams, T. R., Connolly, A. J., Godwin, A. K., Koong, A. C., Wong, A. J. 2012; 31 (24): 2953-2967

  Abstract

  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.

  View details for DOI 10.1038/onc.2011.465

  View details for Web of Science ID 000305388400005

  View details for PubMedID 21986942

• Epidermal Growth Factor Receptor Variant III Contributes to Cancer Stem Cell Phenotypes in Invasive Breast Carcinoma CANCER RESEARCH Del Vecchio, C. A., Jensen, K. C., Nitta, R. T., Shain, A. H., Giacomini, C. P., Wong, A. J. 2012; 72 (10): 2657-2671

  Abstract

  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.

  View details for DOI 10.1158/0008-5472.CAN-11-2656

  View details for Web of Science ID 000307346800020

  View details for PubMedID 22419663

• Immunohistochemical discrimination of wild-type EGFR from EGFRvIII in fixed tumour specimens using anti-EGFR mAbs ICR9 and ICR10 BRITISH JOURNAL OF CANCER Modjtahedi, H., Khelwatty, S. A., Kirk, R. S., Seddon, A. M., Essapen, S., Del Vecchio, C. A., Wong, A. J., Eccles, S. 2012; 106 (5): 883-888

  Abstract

  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.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.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.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.

  View details for DOI 10.1038/bjc.2012.27

  View details for Web of Science ID 000300894300012

  View details for PubMedID 22315050

• Targeting EGF receptor variant III: tumor-specific peptide vaccination for malignant gliomas EXPERT REVIEW OF VACCINES Del Vecchio, C. A., Li, G., Wong, A. J. 2012; 11 (2): 133-144

  Abstract

  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.

  View details for DOI 10.1586/ERV.11.177

  View details for Web of Science ID 000300758700009

  View details for PubMedID 22309662

• Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Monje, M., Mitra, S. S., Freret, M. E., Raveh, T. B., Kim, J., Masek, M., Attema, J. L., Li, G., Haddix, T., Edwards, M. S., Fisher, P. G., Weissman, I. L., Rowitch, D. H., Vogel, H., Wong, A. J., Beachy, P. A. 2011; 108 (11): 4453-4458

  Abstract

  Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

  View details for DOI 10.1073/pnas.1101657108

  View details for Web of Science ID 000288450900040

  View details for PubMedID 21368213

  View details for PubMedCentralID PMC3060250

• The role of the c-Jun N-terminal kinase 2-alpha-isoform in non-small cell lung carcinoma tumorigenesis ONCOGENE Nitta, R. T., Del Vecchio, C. A., Chu, A. H., Mitra, S. S., Godwin, A. K., Wong, A. J. 2011; 30 (2): 234-244

  Abstract

  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.

  View details for DOI 10.1038/onc.2010.414

  View details for Web of Science ID 000286438900011

  View details for PubMedID 20871632

• Rindopepimut, a 14-mer injectable peptide vaccine against EGFRvIII for the potential treatment of glioblastoma multiforme CURRENT OPINION IN MOLECULAR THERAPEUTICS Del Vecchio, C. A., Wong, A. J. 2010; 12 (6): 741-754

  Abstract

  Celldex Therapeutics is developing rindopepimut (CDX-110), a 14-mer injectable peptide vaccine for the potential treatment of glioblastoma multiforme (GBM). Rindopepimut specifically targets a novel junctional epitope of the EGFR deletion mutant EGFRvIII, which is a constitutively active receptor that is expressed in approximately 60 to 70% of patients with GBM. EGFRvIII expression is correlated with worse prognosis and reduced overall survival. Importantly, EGFRvIII is not expressed in normal brain tissue, making it an excellent therapeutic target. Preclinical studies demonstrated lasting tumor regression and increased survival times, as well as efficient generation of EGFRvIII-specific humoral and cellular immune responses, in animals expressing EGFRvIII and vaccinated with rindopepimut. Phase I and II clinical trials in patients with GBM demonstrated significantly increased median time to progression and overall survival time in those vaccinated with rindopepimut compared with matched historical controls. Only limited side effects have been observed in patients. Given these results, rindopepimut is an extremely promising therapy for patients with GBM. Phase I and II clinical trials in patients with GBM were ongoing at the time of publication. In the future, it may be beneficial to combine rindopepimut with other treatment modalities to further prolong survival.

  View details for Web of Science ID 000285308300013

  View details for PubMedID 21154166

• IDENTIFICATION AND TARGETING OF GLIOBLASTOMA STEM CELLS USING EGFRVIII Advances in Inflammatory Bowel Diseases Crohn's and Colitis Foundations National Clinical and Research Conference Wong, A. J., Del Vecchio, C., Mitra, S., Han, S., Holgado-Madruga, M., Gupta, P. OXFORD UNIV PRESS INC. 2010: 123–123

  View details for Web of Science ID 000285082400521

• Development of an EGFRvIII specific recombinant antibody BMC BIOTECHNOLOGY Gupta, P., Han, S., Holgado-Madruga, M., Mitra, S. S., Li, G., Nitta, R. T., Wong, A. J. 2010; 10

  Abstract

  EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 10⁷ M⁻¹ as determined by enzyme-linked immunosorbent assay (ELISA).This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.

  View details for DOI 10.1186/1472-6750-10-72

  View details for Web of Science ID 000283354200001

  View details for PubMedID 20925961

• Pineal Parenchymal Tumor of Intermediate Differentiation: Clinicopathological Report and Analysis of Epidermal Growth Factor Receptor Variant III Expression NEUROSURGERY Li, G., Mitra, S., Karamchandani, J., Edwards, M. S., Wong, A. J. 2010; 66 (5): 963-968

  Abstract

  Epidermal growth factor receptor (EGF) receptor gene amplification is commonly seen in cancer and is the target of many therapies. EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor and has been detected in a large percentage of patients with glioblastoma multiforme but not in normal brain. Therapies targeting EGFRvIII are currently being investigated in clinical and preclinical trials.A 14-year-old girl who presented with headaches was found to have a pineal parenchymal tumor of intermediate differentiation. We review the histopathological properties that led to the diagnosis of this tumor. EGF receptor gene amplification and EGFRvIII expression have not been analyzed in pineal tumors. We investigated EGF receptor gene status and EGFRvIII expression in this patient's tumor.Tumor tissue was obtained and analyzed with flow cytometry, reverse-transcriptase polymerase chain reaction, and Western blot analysis. EGFRvIII was detected by all 3 methods. The tumor was further analyzed by fluorescence in situ hybridization, which did not reveal EGF receptor gene amplification.This is the first report of EGFRvIII expression in a pineal tumor. It is interesting that this variant is detected in the absence of EGF receptor gene amplification. A larger study evaluating the presence of EGFRvIII in pineal tumors is needed.

  View details for DOI 10.1227/01.NEU.0000367726.49003.F1

  View details for Web of Science ID 000276970800026

  View details for PubMedID 20404701

• MEASURING THE CONSTITUTIVE ACTIVATION OF C-JUN N-TERMINAL KINASE ISOFORMS METHODS IN ENZYMOLOGY, VOLUME 484: CONSTITUTIVE ACTIVITY IN RECEPTORS AND OTHER PROTEINS, PART A Nitta, R. T., Badal, S. S., Wong, A. J. 2010; 484: 531-548

  Abstract

  The c-Jun N-terminal kinases (JNK) are important regulators of cell growth, proliferation, and apoptosis. JNKs are typically activated by a sequence of events that include phosphorylation of its T-P-Y motif by an upstream kinase, followed by homodimerization and translocation to the nucleus. Constitutive activation of JNK has been found in a variety of cancers including non-small cell lung carcinomas, gliomas, and mantle cell lymphoma. In vitro studies show that constitutive activation of JNK induces a transformed phenotype in fibroblasts and enhances tumorigenicity in a variety of cell lines. Interestingly, a subset of JNK isoforms was recently found to autoactivate rendering the proteins constitutively active. These constitutively active JNK proteins were found to play a pivotal role in activating transcription factors that increase cellular growth and tumor formation in mice. In this chapter, we describe techniques and methods that have been successfully used to study the three components of JNK activation. Use of these techniques may lead to a better understanding of the components of JNK pathways and how JNK is activated in cancer cells.

  View details for DOI 10.1016/S0076-6879(10)84026-1

  View details for Web of Science ID 000284508500026

  View details for PubMedID 21036249

• The Epidermal Growth Factor Variant III Peptide Vaccine for Treatment of Malignant Gliomas NEUROSURGERY CLINICS OF NORTH AMERICA Li, G., Mitra, S., Wong, A. J. 2010; 21 (1): 87-?

  Abstract

  Epidermal growth factor variant III (EGFRvIII) is the most common alteration of the epidermal growth factor (EGF) receptor found in human tumors. It is commonly expressed in glioblastoma multiforme (GBM), where it was initially identified. This constitutively active mutant receptor leads to unregulated growth, survival, invasion, and angiogenesis in cells that express it. EGFRvIII results from an in-frame deletion of exons 2 to 7 resulting in the fusion of exon 1 to exon 8 of the EGF receptor gene creating a novel glycine at the junction in the extracellular amino terminal domain. The juxtaposition of ordinarily distant amino acids in combination with the glycine that forms at the junction leads to a novel tumor-specific epitope that would make an ideal tumor-specific target. A peptide derived from the EGFRvIII junction can be used as a vaccine to prevent or induce the regression of tumors. This peptide vaccine has now proceeded to phase 1 and 2 clinical trials where it has been highly successful and is now undergoing investigation in a larger human clinical trial for patients who have newly diagnosed GBM. In this article, the authors discuss the preclinical data that led to the human trials and the exciting preliminary data from the clinical trials.

  View details for DOI 10.1016/j.nec.2009.08.004

  View details for Web of Science ID 000278059500009

  View details for PubMedID 19944969

• Targeting brain tumor stem cells using a bispecific antibody directed against CD133+and EGFRvIII 45th Annual Meeting of the American-Society-of-Clinical-Oncology (ASCO) Wong, A., Mitra, S., Gupta, P. AMER SOC CLINICAL ONCOLOGY. 2009

  View details for Web of Science ID 000276606602170

• Constitutive Activity of JNK2 alpha 2 Is Dependent on a Unique Mechanism of MAPK Activation JOURNAL OF BIOLOGICAL CHEMISTRY Nitta, R. T., Chu, A. H., Wong, A. J. 2008; 283 (50): 34935-34945

  Abstract

  c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2alpha2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2alpha2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2alpha2 autophosphorylation and autoactivation. We find that JNK2alpha2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2alpha2 dimerization rendering JNK2alpha2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2alpha2 homodimerization. We also show that a kinase-inactive JNK2alpha2 mutant can interact with and inhibit wild type JNK2alpha2 autophosphorylation, suggesting that JNK2alpha2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2alpha2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2alpha2 activity and that prevention of dimerization may decrease JNK2alpha2 induced tumorigenic phenotypes.

  View details for DOI 10.1074/jbc.M804970200

  View details for Web of Science ID 000261469100050

  View details for PubMedID 18940813

• IDENTIFICATION OF EGFRVIII IN BRAIN TUMOR STEM CELL ANALYSIS SUGGESTS A BISPECIFIC ANTIBODY TARGETING STRATEGY 13th Annual Meeting of the Society-for-Neuro-Oncology (SNO) Mitra, S., Del Vecchio, C., Han, S., Gupta, P., Skirboll, S., Wong, A. OXFORD UNIV PRESS INC. 2008: 899–99

  View details for Web of Science ID 000259854500513

• EGF receptor variant III as a target antigen for tumor immunotherapy EXPERT REVIEW OF VACCINES Li, G., Wong, A. J. 2008; 7 (7): 977-985

  Abstract

  The EGF receptor (EGFR) is the first tyrosine kinase receptor ever cloned and remains at the forefront of targeted therapies against cancer. Currently, there are four US FDA-approved drugs and several more in Phase III studies that target the EGFR. These drugs, while resulting in some dramatic remissions, have not resulted in strong nor consistent improvements in survival. EGFR variant III (EGFRvIII) is the most common variant of the EGFR and is present in many different cancer types but not in normal tissue. It results from the fusion of exon 1 to exon 8 of the EGFR gene, which results in a novel glycine at the junction. This mutant receptor is constitutively active in these tumors and can lead directly to cancer phenotypes due to its oncogenic properties. EGFRvIII is an attractive target antigen for cancer immunotherapy because it is not expressed in normal tissue and because cells producing EGFRvIII have an enhanced capacity for dysregulated growth, survival, invasion and angiogenesis. In this review, we will discuss preclinical and clinical data from studies using EGFRvIII as the target antigen for immunotherapy, with a focus on the potential for greatly improved survival for patients diagnosed with glioblastoma multiforme.

  View details for DOI 10.1586/14760584.7.7.977

  View details for Web of Science ID 000259334600016

  View details for PubMedID 18767947

• The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation JOURNAL OF BIOLOGICAL CHEMISTRY Laramee, M., Chabot, C., Cloutier, M., Stenne, R., Holgado-Madruga, M., Wong, A. J., Royal, I. 2007; 282 (11): 7758-7769

  Abstract

  Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

  View details for DOI 10.1074/jbc.M611327200

  View details for Web of Science ID 000245081000003

  View details for PubMedID 17178724

• A variant epidermal growth factor receptor protein is similarly expressed in benign hyperplastic and carcinomatous prostatic tissues in black and white men. West African journal of medicine Olapade-Olaopa, E. O., Moscatello, D. K., MacKay, E. H., Ogunbiyi, J. O., Shittu, O. B., Terry, T. R., OKEKE, L. I., Wong, A. J., Habib, F. K. 2007; 26 (1): 42-47

  Abstract

  Anti-epidermal growth factor receptor strategies are now established in cancer treatment We have recently described the presence of EGFRvIII (a variant EGFR) in prostatic tumours from UK white men and this is now a target for anti-prostate cancer treatments. However, there has been no report on the expression of this abnormal protein in black men.We determined EGFRvIII expression in sections of normal, benign hyperplastic (BPH) and carcinomatous (CaP) prostatic archival tissues from Nigerian men and UK white men using streptavidin immunohistochemical techniques. The EGFRvIII immunoreactivity was scored visually using a semi-quantitative method and the results compared statistically.EGFRvIII expression increased with increasing malignancy in both study populations (CaP > BPH > Normal p, <0.0001). Furthermore, EGFRvIII expression was similar in both BPH and CaP tissues in black and white men (p, 0.86 and 0.31 respectively).These results demonstrate that EGFRvIII immunoreactivity in prostatic tumours in black men is similar to that in white men. Anti-cancer treatments directed at the EGFRvIII should be equally effective in men from both subpopulations.

  View details for PubMedID 17595991

• c-Jun NH2-terminal kinase 2 alpha 2 promotes the tumorigenicity of human glioblastoma cells CANCER RESEARCH Cui, J., Han, S., Wang, C., Su, W., Harshyne, L., Holgado-Madruga, M., Wong, A. J. 2006; 66 (20): 10024-10031

  Abstract

  c-Jun NH(2)-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 JNK2alpha2 or JNK2beta2. Notably, we found that only JNK2 isoforms possess intrinsic autophosphorylation activity and that JNK2alpha2 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 JNK2alpha2 is expressed in 91% (10 of 11) of glioblastoma tumors, whereas JNK2beta2 is found in only 27% (3 of 11) of tumors. Both JNK2alpha2 and JNK2beta2 mRNAs are expressed in normal brain (3 of 3). Using an antibody specific for JNK2alpha isoforms, we verified that JNK2alpha2 protein is expressed in 88.2% (15 of 17) of glioblastomas, but, interestingly, no JNK2alpha2 protein was found in six normal brain samples. To evaluate biological function, we transfected U87MG cells with green fluorescent protein-tagged versions of JNK1alpha1, JNK2alpha2, and JNK2alpha2APF (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, JNK2alpha2 was found to be the most effective in promoting that phenotype. To identify effectors specifically affected by JNK2alpha2, we analyzed gene expression. Gene profiling showed several genes whose expression was specifically up-regulated by JNK2alpha2 but down-regulated by JNK2alpha2APF, 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 JNK2alpha2 could specifically activate AKT. Our data provides evidence that JNK2alpha2 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.

  View details for DOI 10.1158/0008-5472.CAN-06-0136

  View details for Web of Science ID 000241392700030

  View details for PubMedID 17047065

• beta(1) integrins modulate cell adhesion by regulating insulin-like growth factor-II levels in the microenvironment CANCER RESEARCH Goel, H. L., Moro, L., King, M., Teider, N., CENTRELLA, M., McCarthy, T. L., Holgado-Madruga, M., Wong, A. J., Marra, E., Languino, L. R. 2006; 66 (1): 331-342

  Abstract

  The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C 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, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C 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 beta1 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 beta1C 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 beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.

  View details for DOI 10.1158/0008-5472.CAN-05-2588

  View details for Web of Science ID 000234529500041

  View details for PubMedID 16397247

• Identification of a specific domain responsible for JNK2 alpha 2 autophosphorylation JOURNAL OF BIOLOGICAL CHEMISTRY Cui, J., Holgado-Madruga, M., Su, W. W., Tsuiki, H., Wedegaertner, P., Wong, A. J. 2005; 280 (11): 9913-9920

  Abstract

  c-Jun N-terminal kinases (JNKs) are a group of mitogen-activated protein kinase family members that are important in regulating cell growth, proliferation, and apoptosis. Activation of the JNK pathway has been implicated in the formation of several human tumors. We have previously demonstrated that a 55-kDa JNK isoform is constitutively activated in 86% of human brain tumors and more recently demonstrated that this isoform is either JNK2alpha2 or JNK2beta2. Importantly, we have also found that among the 10 known JNK isoforms, the JNK2 isoforms are unique in their ability to autophosphorylate in vitro and in vivo. This does not require the participation of any upstream kinases and also leads to substrate kinase activity in vitro and in vivo. To clarify the mechanism of JNK2alpha2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1alpha2, which does not have detectable autophosphorylation activity, with portions of JNK2alpha2, which has the strongest autophosphorylation activity. Through in vivo and in vitro kinase assays, we were able to define a domain ranging from amino acids 218 to 226 within JNK2alpha2 that is required for its autophosphorylation. Mutation of JNK2alpha2 to its counterpart of JNK1alpha2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1alpha2 to JNK2alpha2 at this 9-amino acid site enabled JNK1alpha2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2alpha2 activity. One site ranging from amino acids 363 to 382 of JNK2alpha2 is required for efficient c-Jun binding in vitro, and a site ranging from amino acids 383 to 424 enhances autophosphorylation intensity, although it is not required for triggering the autophosphorylation in vitro. These findings have uncovered the regions required for JNK2alpha2 autophosphorylation, and this information could be used as potential targets to block JNK2alpha2 activation.

  View details for DOI 10.1074/jbc.M412165200

  View details for Web of Science ID 000227559600022

  View details for PubMedID 15637069

• Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes CELLULAR MICROBIOLOGY Sun, H., Shen, Y., Dokainish, H., Holgado-Madruga, M., Wong, A., Ireton, K. 2005; 7 (3): 443-457

  Abstract

  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.

  View details for DOI 10.1111/j.1462-5822.2004.00475.x

  View details for Web of Science ID 000226641500013

  View details for PubMedID 15679846

• Alterations in the expression of androgen receptor, wild type-epidermal growth factor receptor and a mutant epidermal growth factor receptor in human prostate cancer. African journal of medicine and medical sciences Olapade-Olaopa, E. O., Moscatello, D. K., MacKay, E. H., Sandhu, D. P., Terry, T. R., Wong, A. J., Habib, F. K. 2004; 33 (3): 245-253

  Abstract

  Prostatic carcinogenesis has been associated with alterations in the expression of the androgen receptor (AR) and the epidermal growth factor receptor (WT-EGFR), and over-expression of the constitutively active variant epidermal growth factor receptor (EGFRvIII). Changes in the expression of AR, WT-EGFR and EGFRvIII were evaluated in serial sections from 26 normal and 26 benign hyperplastic and 50 prostate cancer tissues using specific immunostaining techniques. The loss of AR expression in peri-epithelial stroma as prostatic tissues de-differentiated correlated strongly with the depletion of WT-EGFR and with increasing expression of the EGFRvIII in the adjacent epithelium. In contrast, changes in epithelial AR immunopositivity in these tissues correlated weakly with the changes in normal and variant EGFR levels. This is the first report correlating the changes in the expression of these three proteins in archival material from the different human prostatic tissue histotypes. The loss of expression of proteins that contribute to the regulation of prostatic homeostasis (AR and WT-EGFR) correlates strongly with the expression of a constitutively active variant EGF receptor (EGFRvIII) in human prostate cancer. These changes occur at an early stage of neoplastic transformation and may contribute to the progression of the disease to hormone independence.

  View details for PubMedID 15819472

• Role of the Grb2-associated binder 1/SHP-2 interaction in cell growth and transformation CANCER RESEARCH Holgado-Madruga, M., Wong, A. J. 2004; 64 (6): 2007-2015

  Abstract

  Grb2-associated binder 1 (Gab1) is a docking protein that is tyrosine phosphorylated following the activation of multiple cytokine receptors and receptor tyrosine kinases. Its function then is to recruit and activate multiple signaling molecules. In our previous work, we showed that Gab1 enhances cell growth and induces the transformed phenotype in NIH3T3 cells downstream of the epidermal growth factor (EGF) receptor. In this report, we analyze how it produces these effects. Because SHP-2 is the major binding partner of Gab1, we mutated its binding site in the Gab1 cDNA (Gab1/DeltaSHP-2). This construct was stably overexpressed in NIH3T3 cells (3T3-Gab1/DeltaSHP-2) and in the wild-type Gab1 cDNA (3T3-Gab1) or an empty expression vector (3T3-CTR). Our findings show that after EGF stimulation, Gab1/DeltaSHP-2 has a higher level of tyrosine phosphorylation at early time points than Gab1. Gab1/DeltaSHP-2 recruits more phosphatidylinositol 3'-kinase than Gab1 after EGF triggering, which accounts for a higher and more sustained AKT activation in 3T3-Gab1/DeltaSHP-2 cells relative to 3T3-Gab1 fibroblasts. Moreover, 3T3-Gab1/DeltaSHP-2 cells demonstrate a higher level of extracellular-regulated kinase 1 activation at early time points of EGF stimulation. However, there was an unexpected decrease in c-fos promoter induction in 3T3-Gab1/DeltaSHP-2 cells when compared with 3T3-Gab1 cells. Additionally, the 3T3-Gab1/DeltaSHP-2 cells show a reversion of the transformed phenotype, including fewer morphologic changes, an increase in stress fiber cytoskeletal organization, and a decrease in cell proliferation and anchorage independent growth. These results reveal that the Gab1/SHP-2 interaction is essential for cell growth and transformation but that this must occur through a novel pathway that is independent of extracellular-regulated kinase or AKT. On the basis of its role in growth and transformation, the Gab1/SHP-2 interaction may become an attractive target for the pharmacologic intervention of malignant cell growth.

  View details for Web of Science ID 000220249100019

  View details for PubMedID 15026337

• Gab1 is an integrator of cell death versus cell survival signals in oxidative stress MOLECULAR AND CELLULAR BIOLOGY Holgado-Madruga, M., Wong, A. J. 2003; 23 (13): 4471-4484

  Abstract

  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.

  View details for DOI 10.1128/MCB.23.13.4471-4484.2003

  View details for Web of Science ID 000183848200006

  View details for PubMedID 12808090

• Targeting of cells expressing wild-type EGFR and type-III mutant EGFR (EGFRVIII) by anti-EGFR MAb ICR62: A two-pronged attack for tumour therapy INTERNATIONAL JOURNAL OF CANCER Modjtahedi, H., Moscatello, D. K., Box, G., Green, M., Shotton, C., LAMB, D. J., Reynolds, L. J., Wong, A. J., Dean, C., Thomas, H., Eccles, S. 2003; 105 (2): 273-280

  Abstract

  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.

  View details for DOI 10.1002/ijc.11055

  View details for Web of Science ID 000182359500020

  View details for PubMedID 12673691

• Constitutively active forms of c-Jun NH2-terminal kinase are expressed in primary glial tumors CANCER RESEARCH Tsuiki, H., Tnani, M., Okamoto, I., Kenyon, L. C., Emlet, D. R., Holgado-Madruga, M., Lanham, I. S., Joynes, C. J., Vo, K. T., Wong, A. J. 2003; 63 (1): 250-255

  Abstract

  The c-Jun NH(2)-terminal kinases (JNKs) have a role both in promoting apoptosis and tumorigenesis. The JNKs are encoded by three separate genes (JNK1, 2, and 3), which are spliced alternatively to create 10 JNK isoforms that are either M(r) 55,000 or 46,000 in size. However, the functional significance and distinct role for each splice variant remains unclear. We have noted previously that 86% of primary human glial tumors show activation of almost exclusively the M(r) 55,000 isoforms of JNK. To further study which isoforms are involved, we constructed glutathione S-transferase fusion proteins for all 10 JNK isoforms and examined kinase activity with or without the activating upstream kinase. Surprisingly, five JNK isoforms demonstrate autophosphorylation activity, and in addition, all four JNK2 isoforms (either M(r) 55,000 or 46,000) show a high basal level of substrate kinase activity in the absence of the upstream kinase, especially a M(r) 55,000 JNK2 isoform. Examination revealed autophosphorylation activity at the T-P-Y motif, which is critical for JNK activation, because a mutant lacking the dual phosphorylation sites did not show autophosphorylation or basal kinase activity. Using green fluorescence protein-JNK expression vectors, transient transfection into U87MG cells demonstrates that although the JNK1 isoforms localize predominantly to the cytoplasm, the JNK2 isoforms localize to the nucleus and are phosphorylated, confirming the constitutive activation seen in vitro. We then examined which JNK isoforms are active in glial tumors by performing two-dimensional electrophoresis. This revealed that the M(r) 55,000 isoforms of JNK2 are the principal active JNK isoforms present in tumors. Collectively, these results suggest that these constitutively active JNK isoforms play a significant role in glial tumors. Aside from epidermal growth factor receptor vIII, this is the only other kinase that has been shown to be basally active in glioma. The presence of constitutively active JNK isoforms may have implications for the design of inhibitors of the JNK pathway.

  View details for Web of Science ID 000180290500039

  View details for PubMedID 12517805

• Expression of constitutively activated EGFRvIII in non-small cell lung cancer CANCER SCIENCE Okamoto, I., Kenyon, L. C., Emlet, D. R., Mori, T., Sasaki, J., Hirosako, S., Ichikawa, Y., Kishi, H., Godwin, A. K., Yoshioka, M., Suga, M., Matsumoto, M., Wong, A. J. 2003; 94 (1): 50-56

  Abstract

  The epidermal growth factor receptor (EGFR) variant type III (variously called EGFRvIII, de2-7 EGFR or deltaEGFR) has an in-frame deletion of the extracellular domain and is found in numerous types of human tumors. Since EGFRvIII has been reported to be tumor-specific and has oncogenic potential, it is being investigated as a potential therapeutic target. Because the cell-specific expression of EGFRvIII in lung has not been well documented, we examined the expression of EGFRvIII in 76 non-small cell lung cancers (NSCLCs) and 10 non-neoplastic lung tissues by immunohistochemistry using a new monoclonal antibody specific for this variant receptor. We found a higher incidence (30 of 76, 39%) of enhanced EGFRvIII expression in NSCLC than previously described. Interestingly, the presence of EGFRvIII was also observed in several normal tissue components of lung (e.g., normal bronchial epithelium). Given the high prevalence of EGFRvIII in NSCLC, a newly developed phospho-specific (activated) EGFR antibody was employed for immunohistochemical analysis that permitted visualization of activated EGFR and/or EGFRvIII in tumors. This study presents evidence, for the first time, that EGFRvIII expressed in human tumors is phosphorylated and hence activated. Our results suggest that the sustained activation of EGFRvIII is implicated in the pathogenesis of NSCLC and thus EGFRvIII is a potential therapeutic target in this challenging disease.

  View details for Web of Science ID 000182370000009

  View details for PubMedID 12708474

• Elevated JNK activation contributes to the pathogenesis of human brain tumors ONCOGENE Antonyak, M. A., Kenyon, L. C., Godwin, A. K., James, D. C., Emlet, D. R., Okamoto, I., Tnani, M., Holgado-Madruga, M., Moscatello, D. K., Wong, A. J. 2002; 21 (33): 5038-5046

  Abstract

  The ERK pathway is typically associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. An analogous signaling module, the JNK pathway, has not been shown to be consistently activated by the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines. The function of the JNK pathway in primary tumors is unclear as it has been implicated in both promoting apoptosis and cell growth in vitro, which may be a reflection of the cell lines chosen. Primary human brain tumors frequently show overexpression of the EGF receptor. To clarify the role of JNK in tumorigenesis, we have investigated the role of JNK in a large panel of primary human brain tumors and tumor derived cell lines. Here we present evidence that JNK has a major role in promoting tumorigenesis both in vivo and in vitro. Western blot analysis demonstrated that 86% (18 of 21) of primary brain tumors showed evidence of JNK activation but only 38% (8 of 21) showed evidence of ERK activation. Kinase assays revealed that 77% of brain tumor cell lines activated JNK in response to EGF (7 of 13) or had high levels of basal activity (3 of 13), whereas none of six normal cell lines analysed, including astrocytes, had these properties. Of several growth factors examined, EGF produced the highest level of JNK induction in tumor cell lines and the duration of activation was greater than that seen for ERK. Expression of a dominant-negative (dn) form of JNK potently inhibited EGF mediated anchorage independent growth and protection from cell death in two glial tumor cell lines. These findings demonstrate that enhanced JNK activation is frequently found in primary brain tumors and that this activation contributes to phenotypes related to transformation.

  View details for DOI 10.1038/sj.onc.1205593

  View details for Web of Science ID 000176975900002

  View details for PubMedID 12140754

• Proteolytic cleavage of the CD44 adhesion molecule in multiple human tumors AMERICAN JOURNAL OF PATHOLOGY Okamoto, I., Tsuiki, H., Kenyon, L. C., Godwin, A. K., Emlet, D. R., Holgado-Madruga, M., Lanham, I. S., Joynes, C. J., Vo, K. T., Guha, A., Matsumoto, M., Ushio, Y., Saya, H., Wong, A. J. 2002; 160 (2): 441-447

  Abstract

  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.

  View details for Web of Science ID 000173733500008

  View details for PubMedID 11839564

• Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway JOURNAL OF CELL BIOLOGY Okamoto, I., Kawano, Y., Murakami, D., Sasayama, T., Araki, N., Miki, T., Wong, A. J., Saya, H. 2001; 155 (5): 755-762

  Abstract

  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.

  View details for Web of Science ID 000172459900011

  View details for PubMedID 11714729

• New approaches to the molecular biology, classification, and therapy of nervous system tumors: a workshop of the National Institutes of Health Pathology B study section. American journal of pathology Wong, A. J., Padarathsingh, M., Louis, D. N., Rempel, S., Ladisch, S., Gladson, C. 2001; 159 (5): 1971-1974

  View details for PubMedID 11696458

  View details for PubMedCentralID PMC1867057

• Further characterization of storage-related alterations in immunoreactivity of archival tissue sections and its implications for collaborative multicenter immunohistochemical studies APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY Olapade-Olaopa, E., Ogunbiyi, J. O., MacKay, E. H., Muronda, C. A., Alonge, T. O., Danso, A. P., Moscatello, D. K., Sandhu, D. P., Shittu, O. B., Terry, T. R., Wong, A. J., Habib, F. K. 2001; 9 (3): 261-266

  Abstract

  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.

  View details for Web of Science ID 000170625800011

  View details for PubMedID 11556755

• The Gab1 docking protein links the B cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase JOURNAL OF BIOLOGICAL CHEMISTRY Ingham, R. J., Santos, L., Dang-Lawson, M., Holgado-Madruga, M., Dudek, P., Maroun, C. R., Wong, A. J., Matsuuchi, L., Gold, M. R. 2001; 276 (15): 12257-12265

  Abstract

  B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.

  View details for Web of Science ID 000168081800110

  View details for PubMedID 11278704

• Grb-2-associated binder-1 is involved in insulin-induced egr-1 gene expression through its phosphatidylinositol 3 '-kinase binding site DNA AND CELL BIOLOGY Harada, S., Esch, G. L., Holgado-Madruga, M., Wong, A. J. 2001; 20 (4): 223-229

  Abstract

  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.

  View details for Web of Science ID 000168518800004

  View details for PubMedID 11403719

• The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the Met receptor tyrosine kinase MOLECULAR AND CELLULAR BIOLOGY Maroun, C. R., Naujokas, M. A., Holgado-Madruga, M., Wong, A. J., Park, M. 2000; 20 (22): 8513-8525

  Abstract

  Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

  View details for Web of Science ID 000090094800020

  View details for PubMedID 11046147

• Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer CANCER RESEARCH Tang, C. K., Gong, X. Q., Moscatello, D. K., Wong, A. J., LIPPMAN, M. E. 2000; 60 (11): 3081-3087

  Abstract

  Epidermal growth factor receptor vIII (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the EGFR. Its expression has been detected in gliomas and various other human malignancies. To more fully characterize the function and potential biological role of EGFRvIII in regulating cell proliferation and in tumorigenesis, we transfected EGFRvIII cDNA into a nontumorigenic, interleukin 3 (IL-3)-dependent murine hematopoietic cell line (32D cells). We observed 32D cells expressing high levels of EGFRvIII (32D/EGFRvIII P5) to be capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, the parental cells, 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells, all depended on IL-3 or EGF-like ligands for growth. 32D/EGFRvIII P5 cells subjected to long-term culture conditions in the absence of IL-3 revealed further elevation of EGFRvIII expression levels. These results suggested that the IL-3-independent phenotype is mediated by EGFRvIII. The level of expression is a critical driving force for the IL-3-independent phenotype. Dose-response analysis revealed 32D/EGFRvIII cells to require 500-fold higher concentrations (50 ng/ml) of EGF to further stimulate the EGF-mediated proliferation than in the 32D/EGFR cells (100 pg/ml). Similar effects were also observed in beta-cellulin-mediated proliferation. Moreover, 32D cells expressing high levels of EGFRvIII formed large tumors in nude mice, even when no exogenous EGF ligand was administered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells or low-expressing clone 32D/EGFRvIII C2 cells or the parental 32D cells. The changes of the ligand specificity support the notion for an altered conformation of EGFRvIII to reveal an activated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB. These studies clearly demonstrate that EGFRvIII is capable of transforming a nontumorigenic, IL-3-dependent murine hematopoietic cell line (32D cells) into an IL-3-independent and ligand-independent malignant phenotype in vitro and in vivo. To delineate the biological significance of EGFRvIII in human breast cancer, we expressed EGFRvIII in the MCF-7 human breast cancer cell line. Expression of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. Expression of EGFRvIII in MCF-7 cells also elevated ErbB-2 phosphorylation, presumably through heterodimerization and cross-talk. These MCF-7/EGFRvIII transfectants exhibited an approximately 3-fold increase in colony formation in 1% serum with no significant effect observed at higher percentages of serum. A similar result was also seen in anchorage-dependent assays. Furthermore, EGFRvIII expression significantly enhanced tumorigenicity of MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these results provide the first evidence that EGFRvIII could play a pivotal role in human breast cancer progression.

  View details for Web of Science ID 000087434700055

  View details for PubMedID 10850460

• Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer BRITISH JOURNAL OF CANCER Olapade-Olaopa, E. O., Moscatello, D. K., MacKay, E. H., Horsburgh, T., Sandhu, D. P., Terry, T. R., Wong, A. J., Habib, F. K. 2000; 82 (1): 186-194

  Abstract

  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.

  View details for Web of Science ID 000084335900033

  View details for PubMedID 10638988

• Gab1 mediates neurite outgrowth DNA synthesis, and survival in PC12 cells JOURNAL OF BIOLOGICAL CHEMISTRY Korhonen, J. M., Said, F. A., Wong, A. J., Kaplan, D. R. 1999; 274 (52): 37307-37314

  Abstract

  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.

  View details for Web of Science ID 000084382700068

  View details for PubMedID 10601297

• A conserved inositol phospholipid binding site within the pleckstrin homology domain of the Gab1 docking protein is required for epithelial morphogenesis JOURNAL OF BIOLOGICAL CHEMISTRY Maroun, C. R., Moscatello, D. K., Naujokas, M. A., Holgado-Madruga, M., Wong, A. J., Park, M. 1999; 274 (44): 31719-31726

  Abstract

  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.

  View details for Web of Science ID 000083379400087

  View details for PubMedID 10531383

• Expression of OVCA1, a candidate tumor suppressor, is reduced in tumors and inhibits growth of ovarian cancer cells CANCER RESEARCH Bruening, W., Prowse, A. H., Schultz, D. C., Holgado-Madruga, M., Wong, A., Godwin, A. K. 1999; 59 (19): 4973-4983

  Abstract

  Loss of all or part of one copy of chromosome 17p is very common in ovarian and breast tumors. OVCA1 is a candidate tumor suppressor gene mapping to a highly conserved region on chromosome 17p13.3 that shows frequent loss of heterozygosity in breast and ovarian carcinomas. Western blot analysis of extracts prepared from breast and ovarian carcinomas revealed reduced expression of OVCA1 compared with extracts from normal epithelial cells from these tissues. Subcellular localization studies indicate that OVCA1 is localized to punctate bodies scattered throughout the cell but is primarily clustered around the nucleus. Attempts to create cell lines that stably expressed OVCA1 from the cytomegalovirus promoter were generally unsuccessful in a variety of different cell lines. This reduction of colony formation was quantified in the ovarian cancer cell line A2780, where it was demonstrated that cells transfected with plasmids expressing OVCA1 had a 50-60% reduction in colony number as compared with appropriate controls, and only a few of these clones expressed OVCA1, albeit at low levels. The clones that expressed exogenous OVCA1 were found to have dramatically reduced rates of proliferation. Reduced growth rates correlated with an increased proportion of the cells in the G1 fraction of the cell cycle compared with the parental cell line and decreased levels of cyclin D1. The low levels of cyclin D1 appeared to be caused by an accelerated rate of cyclin D1 degradation. Overexpression of cyclin D1 was able to override OVCA1's suppression of clonal outgrowth. These results suggest that slight alterations in the level of OVCA1, such as would occur after reduction of chromosome 17p13.13 to hemizygosity, may result in cell cycle deregulation and promote tumorigenesis.

  View details for Web of Science ID 000083028800036

  View details for PubMedID 10519411

• The Gab1 PH domain is required for localization of Gab1 at sites of cell-cell contact and epithelial morphogenesis downstream from the Met receptor tyrosine kinase MOLECULAR AND CELLULAR BIOLOGY Maroun, C. R., Holgado-Madruga, M., Royal, I., Naujokas, M. A., Fournier, T. M., Wong, A. J., Park, M. 1999; 19 (3): 1784-1799

  Abstract

  Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.

  View details for Web of Science ID 000078697900017

  View details for PubMedID 10022866

• 1998 Neurotumor Club. March 30, 1998, New Orleans, La. Neuro-oncology Gladson, C. L., Guha, A., Wong, A. J. 1999; 1 (1): 30-32

  View details for PubMedID 11550299

  View details for PubMedCentralID PMC1919465

• The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor JOURNAL OF BIOLOGICAL CHEMISTRY Ingham, R. J., Holgado-Madruga, M., Siu, C., Wong, A. J., Gold, M. R. 1998; 273 (46): 30630-30637

  Abstract

  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.

  View details for Web of Science ID 000077008100082

  View details for PubMedID 9804835

• Determination of Gab1 (Grb2-associated binder-1) interaction with insulin receptor-signaling molecules MOLECULAR ENDOCRINOLOGY Rocchi, S., Tartare-Deckert, S., Murdaca, J., Holgado-Madruga, M., Wong, A. J., Van Obberghen, E. 1998; 12 (7): 914-923

  Abstract

  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.

  View details for Web of Science ID 000074476700002

  View details for PubMedID 9658397

• Constitutive activation of c-Jun N-terminal kinase by a mutant epidermal growth factor receptor JOURNAL OF BIOLOGICAL CHEMISTRY Antonyak, M. A., Moscatello, D. K., Wong, A. J. 1998; 273 (5): 2817-2822

  Abstract

  Epidermal growth factor receptor (EGF) variant type III (EGFRvIII) is a constitutively active, naturally occurring mutation of the EGF receptor that is found in many types of human tumors. When overexpressed in NIH3T3 fibroblasts, EGFRvIII induces transformation by enhancing cell growth and reducing apoptosis. Analysis of downstream signaling pathways has revealed that extracellular signal-regulated kinase activity is down-regulated, raising doubt as to the significance of this pathway in promoting transformation. We investigated whether the c-Jun N-terminal kinase (JNK) pathway was affected by EGFRvIII. NIH3T3 cells expressing EGFRvIII exhibited a high basal level of JNK activity, which was not present in cells overexpressing the normal EGF receptor. Treatment of cells overexpressing EGFRvIII with inhibitors of the EGF receptor or phosphatidylinositol 3-kinase resulted in the down-regulation of JNK activity. Furthermore, the down-regulation of JNK activity was associated with a loss of properties related to transformation, and there was no evidence for JNK activity in the promotion of apoptosis in these cells. These findings implicate constitutive activation of the JNK pathway in transformation by EGFRvIII.

  View details for Web of Science ID 000071736600045

  View details for PubMedID 9446590

• Decorin suppresses tumor cell growth by activating the epidermal growth factor receptor JOURNAL OF CLINICAL INVESTIGATION Moscatello, D. K., Santra, M., Mann, D. M., MCQUILLAN, D. J., Wong, A. J., Iozzo, R. V. 1998; 101 (2): 406-412

  Abstract

  Decorin, a small leucine-rich proteoglycan, is capable of suppressing the growth of various tumor cell lines when expressed ectopically. In this report, we investigated the biochemical mechanism by which decorin inhibits cell cycle progression. In A431 squamous carcinoma cells, decorin proteoglycan or protein core induced a marked growth suppression, when either exogenously added or endogenously produced by a transgene. Decorin caused rapid phosphorylation of the EGF receptor and a concurrent activation of mitogen-activated protein (MAP) kinase signal pathway. This led to a protracted induction of endogenous p21, a potent inhibitor of cyclin-dependent kinases, and ultimate cell cycle arrest. Biglycan, a related proteoglycan, had no effect. Moreover, decorin activated the EGF receptor/MAP kinase/ p21 axis in cell lines of various histogenetic backgrounds. These results provide the first evidence that EGF and decorin converge functionally to regulate the cell cycle through activation of a common pathway which ultimately leads to growth suppression.

  View details for Web of Science ID 000071668000016

  View details for PubMedID 9435313

• Constitutive activation of phosphatidylinositol 3-kinase by a naturally occurring mutant epidermal growth factor receptor JOURNAL OF BIOLOGICAL CHEMISTRY Moscatello, D. K., Holgado-Madruga, M., Emlet, D. R., Montgomery, R. B., Wong, A. J. 1998; 273 (1): 200-206

  Abstract

  The most frequently found alteration of the epidermal growth factor receptor (EGFR) in human tumors is a deletion of exons 2-7. This receptor, termed EGFRvIII, can transform NIH 3T3 cells, and the frequent expression of this variant implies that it confers a selective advantage upon tumor cells in vivo. Although EGFRvIII is a constitutively activated tyrosine kinase, there is no increase in Ras.GTP levels and low levels of mitogen-activated protein kinase activity in NIH 3T3 cells expressing this variant. We investigated whether phosphatidylinositol (PI) 3-kinase was an effector in transformation by the EGFRvIII. High levels of PI 3-kinase activity were constitutively present in EGFRvIII-transformed cells and were dependent upon the kinase activity of the receptor. While mitogen-activated protein kinase activity was quickly down-regulated to basal levels after 12 h of continuous EGFR activation, there was a 3-fold increase in PI 3-kinase activity in cells expressing normal EGFR and an 8-fold increase in cells expressing EGFRvIII after 48 h. This increased activity may reflect enhanced binding to EGFRvIII and the presence of novel PI 3-kinase isoforms. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 blocked both anchorage-independent growth and growth in low serum media and also resulted in morphological reversion of EGFRvIII-transformed cells. These results support an essential role for PI 3-kinase in transformation by this EGFR variant.

  View details for Web of Science ID 000071295600031

  View details for PubMedID 9417065

• Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA HOLGADOMADRUGA, M., Moscatello, D. K., Emlet, D. R., Dieterich, R., Wong, A. J. 1997; 94 (23): 12419-12424

  Abstract

  Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.

  View details for Web of Science ID A1997YF39300033

  View details for PubMedID 9356464

• Association of the multisubstrate docking protein Gab1 with the hepatocyte growth factor receptor requires a functional Grb2 binding site involving tyrosine 1356 JOURNAL OF BIOLOGICAL CHEMISTRY Nguyen, L., HOLGADOMADRUGA, M., Maroun, C., Fixman, E. D., Kamikura, D., Fournier, T., Charest, A., Tremblay, M. L., Wong, A. J., Park, M. 1997; 272 (33): 20811-20819

  Abstract

  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.

  View details for Web of Science ID A1997XR22100076

  View details for PubMedID 9252406

• Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Gab1 JOURNAL OF BIOLOGICAL CHEMISTRY Fixman, E. D., HOLGADOMADRUGA, M., Nguyen, L., Kamikura, D. M., Fournier, T. M., Wong, A. J., Park, M. 1997; 272 (32): 20167-20172

  Abstract

  The Tpr-Met oncoprotein consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. Tpr-Met is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement and has constitutive kinase activity. We have previously demonstrated that a single carboxyl-terminal tyrosine residue, Tyr489, is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and forms a multisubstrate binding site for Grb2, phosphatidylinositol 3' kinase, phospholipase Cgamma, SHP2, and an unknown protein of 110 kDa. A mutant Tpr-Met protein that selectively fails to bind Grb2 has reduced transforming activity, implicating pathways downstream of Grb2 in Tpr-Met mediated cell transformation. We show here that the 110-kDa Tpr-Met substrate corresponds to the recently identified Grb2-associated protein, Gab1. Moreover, we show that tyrosine phosphorylation of the Cbl protooncogene product as well as Gab1 required Tyr489 and correlated with the ability of Tpr-Met to associate with Grb2 and to transform cells, providing evidence that pathways downstream of Gab1 and/or Cbl may play a role in Tpr-Met-mediated cell transformation.

  View details for Web of Science ID A1997XQ05900079

  View details for PubMedID 9242692

• A naturally occurring mutant human epidermal growth factor receptor as a target for peptide vaccine immunotherapy of tumors CANCER RESEARCH Moscatello, D. K., Ramirez, G., Wong, A. J. 1997; 57 (8): 1419-1424

  Abstract

  The type III EGF receptor (EGFRvIII) is the result of an in-frame deletion from nucleotides 275 to 1075 in the EGF receptor cDNA sequence creating a novel epitope at the fusion junction. This spontaneously occurring alteration is found in a high percentage of primary human brain, breast, lung and ovarian tumors. We have explored whether a peptide derived from the fusion junction could serve as the basis for an antitumor vaccine. Preimmunization of mice with this peptide substantially inhibited tumor formation by cells expressing EGFRvIII. Tumor cell inoculation followed by immunization could also enhance the regression of existing tumors. Antibody production was elicited in animals that was highly specific for the novel epitope and also a CTL response that was mediated by CD8+ T lymphocytes. The alteration present in EGFRvIII could serve as the basis for an antitumor vaccine with potentially wide application in humans.

  View details for Web of Science ID A1997WT72300005

  View details for PubMedID 9108438

• Epidermal growth factor receptor stimulation of diacylglycerol kinase BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Montgomery, R. B., Moscatello, D. K., Wong, A. J., Stahl, W. L. 1997; 232 (1): 111-116

  Abstract

  The epidermal growth factor receptor (EGFR) activates formation of the phospholipid signal messenger phosphatidic acid (PA) on ligand binding. We explored the effects of chronic EGF stimulation on cellular PA in NIH3T3 cells expressing intact EGFR a mutant EGFR (EGFRvIII). The presence of EGFRvIII increased PA levels to twice those induced by chronic EGFR activation. Fatty acid methyl ester analysis revealed a marked increase in oleic acid containing PA. No apparent increase in phospholipase D (PLD) activity was detected, and diacylglycerol (DAG) kinase assays demonstrated a marked preference for dioleoyl DAG in the presence of activated EGFR or EGFRvIII. Levels of PA which were lower than would be predicted by DAG kinase activation are explained by increased phosphatidate phosphohydrolase activity. Specific inhibitors of EGFR kinase and DAG kinase suppressed DAG kinase activation and PA production by EGFRvIII. EGFR kinase activation by chronic exposure to ligand or by deletional mutation stimulates formation of a specific form of signalling PA.

  View details for Web of Science ID A1997WN21300023

  View details for PubMedID 9125112

• Subsets of epidermal growth factor receptors during activation and endocytosis JOURNAL OF BIOLOGICAL CHEMISTRY Emlet, D. R., Moscatello, D. K., Ludlow, L. B., Wong, A. J. 1997; 272 (7): 4079-4086

  Abstract

  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.

  View details for Web of Science ID A1997WH01900033

  View details for PubMedID 9020117

• The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68 ONCOGENE Lawe, D. C., Hahn, C., Wong, A. J. 1997; 14 (2): 223-231

  Abstract

  SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by tyrosine kinases. Nck is a ubiquitously expressed adaptor protein whose function has been enigmatic. We performed confocal microscopy to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck was identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. Western blot analysis of nuclear and cytosolic fractions confirmed that there was no translocation in response to growth factor and that tyrosine phosphorylation was specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains revealed differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both nonmitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm.

  View details for Web of Science ID A1997WC81800010

  View details for PubMedID 9010224

• Transformation and altered signal transduction by a naturally occurring mutant EGF receptor ONCOGENE Moscatello, D. K., Montgomery, R. B., Sundareshan, P., McDanel, H., Wong, M. Y., Wong, A. J. 1996; 13 (1): 85-96

  Abstract

  An amino-truncated variant form of the epidermal growth factor receptor (EGFRvIII) has been identified in human brain, breast, lung and ovarian tumors. We have found that overexpression of this mutant EGF receptor in NIH3T3 cells results in transformation as a result of the activation of the receptor kinase via ligand-independent dimerization. Transformation was correlated with tyrosine phosphorylation of only a subset of the proteins observed in cells overexpressing the normal EGF receptor. This suggested that further studies on cells expressing the EGFRvIII might provide insights into the pathways most relevant to transformation. In clones expressing high levels of mutant EGF receptor, the levels of both Grb2 and SHC were decreased. Despite this decrease, much of the endogenous Grb2 immunoprecipitated with EGFRvIII. Interestingly, no increase in ras-GTP loading was found in clones expressing the EGFRvIII and MAP kinase assays indicated only a small increase in activity. These results indicate that high-level expression of the EGFRvIII induces down-regulation of the ras-MAP kinase pathway and that other components involved in EGF receptor signal transduction may play a greater role in neoplastic transformation by the EGFRvIII.

  View details for Web of Science ID A1996UX31900010

  View details for PubMedID 8700557

• Identification of two candidate tumor suppressor genes on chromosome 17p13.3 CANCER RESEARCH Schultz, D. C., Vanderveer, L., Berman, D. B., HAMILTON, T. C., Wong, A. J., Godwin, A. K. 1996; 56 (9): 1997-2002

  Abstract

  A second tumor suppressor locus on 17p that is distinct from TP53 has been identified in brain, breast, lung, and ovarian tumors. Using allelic loss mapping and positional cloning methods, we have recently identified two novel genes, which we refer to as OVCA1 and OVCA2, that map to 17p13.3. The two genes are ubiquitously expressed and encode proteins of 443 and 227 amino acids, respectively, with no known functional motifs. Sequence comparison of OVCA1 and OVCA2 revealed extensive sequence identity and similarity to hypothetical proteins from Saccharomyces cerevisiae, Caenorhabditis elegans, and Rattus species. Northern blot analysis reveals that OVCA1 and OVCA2 mRNA were expressed in normal surface epithelial cells of the ovary, but the level of this transcript is significantly reduced or is undetectable in 92% (11/12) of the ovarian tumors and tumor cell lines analyzed. The location, high degree of amino acid conservation, and reduced expression in ovarian tumors and tumor cell lines suggest that decreased expression of these two genes contributes to ovarian tumorigenesis and should be considered candidate tumor suppressor genes.

  View details for Web of Science ID A1996UG75600010

  View details for PubMedID 8616839

• A Grb2-associated docking protein in EGF- and insulin-receptor signalling NATURE HOLGADOMADRUGA, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K., Wong, A. J. 1996; 379 (6565): 560-564

  Abstract

  The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.

  View details for Web of Science ID A1996TU69300056

  View details for PubMedID 8596638

• Differential modulation of mitogen-activated protein (MAP) kinase extracellular signal-related kinase kinase and MAP kinase activities by a mutant epidermal growth factor receptor JOURNAL OF BIOLOGICAL CHEMISTRY Montgomery, R. B., Moscatello, D. K., Wong, A. J., Cooper, J. A., Stahl, W. L. 1995; 270 (51): 30562-30566

  Abstract

  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 (EGFR-vIII) 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. EGFR-vIII 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 EGFR-vIII 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.

  View details for Web of Science ID A1995TL67500054

  View details for PubMedID 8530489

• FREQUENT EXPRESSION OF A MUTANT EPIDERMAL GROWTH-FACTOR RECEPTOR IN MULTIPLE HUMAN TUMORS CANCER RESEARCH Moscatello, D. K., HOLGADOMADRUGA, M., Godwin, A. K., Ramirez, G., Gunn, G., Zoltick, P. W., Biegel, J. A., Hayes, R. L., Wong, A. J. 1995; 55 (23): 5536-5539

  Abstract

  The epidermal growth factor receptor has received much interest as a target for various antineoplastic agents, but a complication is that many normal tissues also express this receptor. We have previously identified in human glial tumors an 801-bp in-frame deletion within the epidermal growth factor receptor gene that created a novel epitope at the junction. By using Western blot assays with a mutant-specific antibody as a rapid and sensitive means for detecting this alteration in primary human tumors, it was found that 57% (26 of 46) of high-grade and 86% (6 of 7) of low-grade glial tumors, but not normal brain, express this protein. This altered receptor was also present in 66% (4 of 6) of pediatric gliomas and 86% (6 of 7) of medulloblastomas, 78% (21 of 27) of breast carcinomas, and 73% (24 of 32) of ovarian carcinomas. The fact that this receptor is frequently found in tumors but not in normal tissue makes it an attractive candidate for various antitumor strategies.

  View details for Web of Science ID A1995TG21100012

  View details for PubMedID 7585629

• TUMOR-SPECIFIC ANTIEPIDERMAL GROWTH-FACTOR RECEPTOR VARIANT-III MONOCLONAL-ANTIBODIES - USE OF THE TYRAMINE-CELLOBIOSE RADIOIODINATION METHOD ENHANCES CELLULAR RETENTION AND UPTAKE IN TUMOR XENOGRAFTS CANCER RESEARCH Reist, C. J., Archer, G. E., Kurpad, S. N., Wikstrand, C. J., Vaidyanathan, G., Willingham, M. C., Moscatello, D. K., Wong, A. J., Bigner, D. D., Zalutsky, M. R. 1995; 55 (19): 4375-4382

  Abstract

  Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene are characteristics of many types of tumors. One class of EGFR mutations, EGFRvIII, is characterized by an in-frame deletion resulting in a truncated external domain of the receptor. EGFRvIII was first identified in a subset of gliomas and has since been found in some non-small cell lung carcinomas and breast carcinomas. mAbs specific for this variant form of EGFR but unreactive with the wild-type EGFR have been reported from our laboratory. This study further characterizes three of these antibodies. We determined, via radiolabeling techniques and immunofluorescence microscopy, that, after cell binding in vitro, the anti-EGFRvIII-specific mAbs internalize at 37 degrees C. Furthermore, subsequent to internalization, the antibodies were processed intracellularly, presumably by lysosomal degradation. We also examined the use of an alternative radiolabeling procedure that uses nonmetabolizable radio-iodinated tyramine cellobiose. Our results show that the tyramine cellobiose labeling method allows for greater tumor cell retention of radiolabel in vitro (76% for tyramine cellobiose and 27% for Iodo-Gen after 24 h). Paired-label biodistribution studies in athymic mice indicate that anti-EGFRvIII mAb L8A4 localizes specifically to EGFRvIII-expressing tumor xenografts with a maximum of 34.3 +/- 7.6% injected dose/g when labeled using tyramine cellobiose compared with a maximum of 14.9 +/- 4.3% injected dose/g using Iodo-Gen; similar results were obtained with mAb H10. These results suggest that the anti-EGFRvIII mAbs may serve as potential carriers for radioconjugate- and immunotoxin-based therapies for tumors expressing EGFRvIII.

  View details for Web of Science ID A1995RX11400032

  View details for PubMedID 7671250

• MONOCLONAL-ANTIBODIES AGAINST EGFRVIII ARE TUMOR-SPECIFIC AND REACT WITH BREAST AND LUNG CARCINOMAS AND MALIGNANT GLIOMAS CANCER RESEARCH Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey, P. A., Kurpad, S. N., McLendon, R. E., Moscatello, D., Pegram, C. N., Reist, C. J., Traweek, S. T., Wong, A. J., Zalutsky, M. R., Bigner, D. D. 1995; 55 (14): 3140-3148

  Abstract

  Despite molecular biological advances in understanding human cancers, translation into therapy has been less forthcoming; targeting neoplastic cells still requires that tumor-specific markers, preferably those on the cell surface, be identified. The epidermal growth factor receptor (EGFR) exists in a deletion-mutant form, EGFRvIII, which has been identified by genetic and immunological means in a subset of gliomas and non-small cell lung carcinomas. Specific polyvalent antisera to the extracellular portion of the variant were readily induced, but immunization using a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We report here five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic peptide and the intact variant in different formats that maintained secondary and tertiary conformation. These mAbs identify the EGFRvIII on the cell surface with relatively high affinity (KA range, 0.13 to 2.5 x 10(9) M-1) by live cell Scatchard analysis. These mAbs are specific for EGFRvIII as determined by RIA, ELISA, Western blot, analytical flow cytometry, autophosphorylation, and immunohistochemistry. Isolating specific mAbs enabled us to analyze normal and neoplastic human tissue and establish that EGFRvIII is truly tumor specific for subsets of breast carcinomas and for previously reported non-small cell lung carcinomas and gliomas. Also, this receptor is not expressed by any normal human tissues thus far examined, including elements of the peripheral, central nervous, and lymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII positivity in gliomas than previously described and identified an EGFRvIII-positive subset of breast tumors; also, we observed that the EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumors expressing this epitope. Our observations strongly warrant development of this mAb-antigen system as therapy for breast, lung, and central nervous system tumors.

  View details for Web of Science ID A1995RH80200034

  View details for PubMedID 7606735

• THE MOLECULAR-BIOLOGY AND MOLECULAR-GENETICS OF ASTROCYTIC NEOPLASMS SEMINARS IN ONCOLOGY Wong, A. J., Zoltick, P. W., Moscatello, D. K. 1994; 21 (2): 139-148

  View details for Web of Science ID A1994NG63700003

  View details for PubMedID 8153660

• POLYMORPHISM AT CODON-36 OF THE P53 GENE ONCOGENE Felix, C. A., Brown, D. L., MITSUDOMI, T., IKAGAKI, N., Wong, A., Wasserman, R., Womer, R. B., Biegel, J. A. 1994; 9 (1): 327-328

  Abstract

  A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.

  View details for Web of Science ID A1994MW24700041

  View details for PubMedID 8302598

• ONCOGENES AND SIGNAL-TRANSDUCTION HOSPITAL PRACTICE Wong, A. J., Croce, C. M. 1993; 28 (7): 128-?

  Abstract

  Cancer biologists now have a unified concept to guide their search for specific genetic abnormalities and new therapeutic approaches: The genes and proteins that participate in the conversion of normal into malignant cells are also involved in the key process that converts extracellular signals into intracellular events that culminate in division and growth.

  View details for Web of Science ID A1993LM16800018

  View details for PubMedID 8325909

• EXPRESSION OF MUTATED EPIDERMAL GROWTH-FACTOR RECEPTOR BY NONSMALL CELL LUNG CARCINOMAS CANCER RESEARCH DEPALAZZO, I. E., ADAMS, G. P., Sundareshan, P., Wong, A. J., Testa, J. R., Bigner, D. D., Weiner, L. M. 1993; 53 (14): 3217-3220

  Abstract

  The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.

  View details for Web of Science ID A1993LM67700001

  View details for PubMedID 8391918

• STRUCTURAL ALTERATIONS OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR GENE IN HUMAN GLIOMAS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wong, A. J., Ruppert, J. M., Bigner, S. H., GRZESCHIK, C. H., Humphrey, P. A., BIGNER, D. S., VOGELSTEIN, B. 1992; 89 (7): 2965-2969

  Abstract

  The epidermal growth factor receptor (EGFR) gene is amplified in 40% of malignant gliomas, and the amplified genes are frequently rearranged. We have characterized the genetic alterations associated with these rearrangements in five malignant gliomas. In one tumor the rearrangement resulted in the deletion of most of the extracytoplasmic domain of the receptor, resulting in a hybrid mRNA between new sequences and the truncated EGFR sequence. The predicted amino acid sequence of the protein from this tumor was remarkably similar to that described for several viral erbB oncogenes. Four other tumors were noted to have internal deletions of the EGFR gene. These rearrangements brought about in-frame deletions affecting either of two cysteine-rich domains in the extracytoplasmic portion of the molecule. The clonal nature of these alterations, and the fact that identical alterations were seen in more than one tumor, suggests a role for these mutant receptor proteins in tumorigenesis. Further, these studies document the existence of tumor-specific cell surface molecules resulting from somatic mutation.

  View details for Web of Science ID A1992HL81600094

  View details for PubMedID 1557402

• DELETION-MUTANT EPIDERMAL GROWTH-FACTOR RECEPTOR IN HUMAN GLIOMAS - EFFECT OF TYPE-II MUTATION ON RECEPTOR FUNCTION BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Humphrey, P. A., Gangarosa, L. M., Wong, A. J., Archer, G. E., LUNDJOHANSEN, M., Bjerkvig, R., Laerum, O. D., Friedman, H. S., Bigner, D. D. 1991; 178 (3): 1413-1420

  Abstract

  Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.

  View details for Web of Science ID A1991GB27300090

  View details for PubMedID 1678600

• CHARACTERIZATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR IN HUMAN GLIOMA CELL-LINES AND XENOGRAFTS CANCER RESEARCH Bigner, S. H., Humphrey, P. A., Wong, A. J., VOGELSTEIN, B., Mark, J., Friedman, H. S., Bigner, D. D. 1990; 50 (24): 8017-8022

  Abstract

  Both permanent cultured cell lines and athymic mouse xenografts were established from two human glioblastomas. Biopsies from D-245 MG and D-270 MG contained amplified and rearranged epidermal growth factor receptor (EGFR) genes. Although the gene amplification and rearrangement seen originally was maintained in the xenografts, cultured cell lines established from these biopsies lost the amplified rearranged genes in vitro. Analysis of these cell lines and 11 additional permanent human glioma cell lines with normal EGFR gene copy number showed from 2.7 x 10(3) to 4.1 x 10(5) high affinity EGFRs/cell by radioreceptor assay. The RNase A protection assay showed minimal differences in the quantity of EGFR mRNA among the 13 glioma lines, while the D-245 MG and D-270 MG xenografts expressed approximately 10-20 times as much EGFR mRNA as the corresponding cell lines. Immunoprecipitation of EGFR from these lines, including D-245 MG and D-270 MG, demonstrated only the intact Mr 170,000 Da form, while truncated Mr 145,000 Da and 100,000 Da EGFR proteins were immunoprecipitated from the D-270 MG and D-245 MG xenografts, respectively. These studies demonstrate that gliomas with amplification of the EGFR gene are capable of establishing in culture but that the amplified rearranged genes are not maintained. Possible explanations are that the abnormal genes are lost during serial passage or that the cells with amplified rearranged genes only represent a minor subpopulation of cells, which are unable to grow in culture. In either case, these observations suggest that high expression and structural abnormalities of EGFR proteins generated by amplification and rearrangement of the EGFR gene provide a growth advantage for gliomas in vivo but not in vitro.

  View details for Web of Science ID A1990EM16500051

  View details for PubMedID 2253244

• ANTISYNTHETIC PEPTIDE ANTIBODY REACTING AT THE FUSION JUNCTION OF DELETION MUTANT EPIDERMAL GROWTH-FACTOR RECEPTORS IN HUMAN GLIOBLASTOMA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Humphrey, P. A., Wong, A. J., VOGELSTEIN, B., Zalutsky, M. R., Fuller, G. N., Archer, G. E., Friedman, H. S., Kwatra, M. M., Bigner, S. H., Bigner, D. D. 1990; 87 (11): 4207-4211

  Abstract

  We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

  View details for Web of Science ID A1990DG09200039

  View details for PubMedID 1693434

• THE GLI-KRUPPEL FAMILY OF HUMAN GENES MOLECULAR AND CELLULAR BIOLOGY Ruppert, J. M., KINZLER, K. W., Wong, A. J., Bigner, S. H., Kao, F. T., Law, M. L., Seuanez, H. N., OBrien, S. J., VOGELSTEIN, B. 1988; 8 (8): 3104-3113

  Abstract

  Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.

  View details for Web of Science ID A1988P431500014

  View details for PubMedID 2850480

• GENE AMPLIFICATION IN MALIGNANT HUMAN GLIOMAS - CLINICAL AND HISTOPATHOLOGIC ASPECTS JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY Bigner, S. H., Burger, P. C., Wong, A. J., Werner, M. H., Hamilton, S. R., Muhlbaier, L. H., VOGELSTEIN, B., Bigner, D. D. 1988; 47 (3): 191-205

  Abstract

  Gene amplification occurs in 45-50% of malignant human gliomas (MHG). In the present study, 64 genetically characterized gliomas were evaluated to determine if tumors with amplification of the epidermal growth factor receptor (EGFR), N-myc, c-myc, or gli genes had distinctive histopathologic features. There was no significant difference in age (p = 0.10) or gender (p = 0.78) between patients whose tumors contained amplified genes and those whose tumors did not exhibit this characteristic. Although the patients with amplified genes in their tumors survived slightly longer than patients whose tumors had no detectable gene amplification, these differences were not statistically significant (p = 0.21). The 28 tumors with amplification included 24/48 (50%) glioblastoma multiforme, 2/6 (33%) anaplastic astrocytomas and 2/5 (40%) gliosarcomas. No amplification was seen in one oligodendroglioma, three anaplastic mixed gliomas or one giant cell glioblastoma multiforme. Necrosis and endothelial proliferation were equally prevalent among tumors with and without amplification. Comparison of tumors with gene amplification and tumors without this characteristic revealed similar distributions of most morphologic cells types. Although prominent perivascular lymphocytic infiltrates were more frequent in tumors without amplification, this association was of borderline significance statistically. In situ hybridization of tumors with amplification using an EGFR mRNA probe showed intense labeling of different neoplastic cell types including fibrillary and protoplasmic astrocytes, gemistocytes, anaplastic cells, and multinucleated giant cells. Non-neoplastic cells such as hyperplastic endothelium within the tumors did not express detectable EGFR mRNA. These studies demonstrate that (a) cells with quite different morphology within the same tumor can contain the same genetic alteration; (b) tumors of identical histological appearance may have arisen and evolved by different molecular mechanisms; and (c) molecular analyses are necessary to evaluate gene amplification in MHG since this characteristic cannot be accurately predicted by the morphologic or clinical criteria used in this study.

  View details for Web of Science ID A1988N498700001

  View details for PubMedID 3367154

• AMPLIFICATION AND EXPRESSION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR GENE IN HUMAN GLIOMA XENOGRAFTS CANCER RESEARCH Humphrey, P. A., Wong, A. J., VOGELSTEIN, B., Friedman, H. S., Werner, M. H., Bigner, D. D., Bigner, S. H. 1988; 48 (8): 2231-2238

  Abstract

  Xenografts from eight malignant human gliomas were established in athymic mice and were used to study amplification and expression of the epidermal growth factor receptor (EGFR) gene. Tissue identity between biopsy and xenografts was confirmed by karyotypic profiles, which showed that each glioma xenograft retained structural abnormalities, including double minute chromosomes, present in the parent glioma. EGFR gene amplification was found in six of the eight glioma biopsies and their corresponding xenografts. Expression of the EGFR gene was measured by Scatchard analysis, affinity reactions, immunoprecipitations, Western immunoblots, and immunocytochemistry; significant expression of the EGFR gene was only detectable in xenografts with EGFR gene amplification. Moreover, five of the six xenografts with EGFR gene amplification demonstrated structural alterations of the EGFR gene, which was associated with low-molecular-weight EGFR proteins. These xenografts represent an excellent tissue source and in vivo model system for characterizing the epidermal growth factor receptor in malignant human gliomas.

  View details for Web of Science ID A1988M876900042

  View details for PubMedID 3258189

• RELATIONSHIP BETWEEN GENE AMPLIFICATION AND CHROMOSOMAL DEVIATIONS IN MALIGNANT HUMAN GLIOMAS CANCER GENETICS AND CYTOGENETICS Bigner, S. H., Wong, A. J., Mark, J., Muhlbaier, L. H., KINZLER, K. W., VOGELSTEIN, B., Bigner, D. D. 1987; 29 (1): 165-170

  Abstract

  Biopsies of 33 malignant human gliomas were karyotyped and evaluated for amplification (more than eight gene copies per cell) of the epidermal growth factor receptor (EGFR), N-myc, c-myc, and gli genes by Southern blot analysis. Fifteen of 33 tumors showed amplification of EGFR, none had amplified c-myc, one tumor had amplified N-myc, and one had amplification of gli. Thirteen of the 16 (81%) evaluable tumors with gene amplification contained double minutes (DM), and only four of 16 (25%) tumors without demonstrable amplification contained these structures. Polysomy for chromosome #7, in contrast, occurred in 58% of tumors with EGFR amplification and 53% of tumors without amplification of the gene. Structural abnormalities of 7p occurred in two tumors with EGFR amplification and in one tumor without amplification of this gene. These studies suggest that DM are the usual locus for amplified genes (usually EGFR) in human glioma biopsies, but that structural abnormalities of 7p may be associated with EGFR amplification in a small proportion of these tumors. The presence of polysomy 7, however, probably is unrelated to amplification of the EGFR gene.

  View details for Web of Science ID A1987K678300021

  View details for PubMedID 3478127

• INCREASED EXPRESSION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR GENE IN MALIGNANT GLIOMAS IS INVARIABLY ASSOCIATED WITH GENE AMPLIFICATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wong, A. J., Bigner, S. H., Bigner, D. D., KINZLER, K. W., Hamilton, S. R., VOGELSTEIN, B. 1987; 84 (19): 6899-6903

  Abstract

  Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.

  View details for Web of Science ID A1987K314300061

  View details for PubMedID 3477813

• IDENTIFICATION OF AN AMPLIFIED, HIGHLY EXPRESSED GENE IN A HUMAN GLIOMA SCIENCE KINZLER, K. W., Bigner, S. H., Bigner, D. D., Trent, J. M., Law, M. L., OBrien, S. J., Wong, A. J., VOGELSTEIN, B. 1987; 236 (4797): 70-73

  Abstract

  A gene, termed gli, was identified that is amplified more than 50-fold in a malignant glioma. The gene is expressed at high levels in the original tumor and its derived cell line and is located at chromosome 12 position (q13 to q14.3). The gli gene is a member of a select group of cellular genes that are genetically altered in primary human tumors.

  View details for Web of Science ID A1987G661400029

  View details for PubMedID 3563490

• GENE AMPLIFICATION OF C-MYC AND N-MYC IN SMALL CELL-CARCINOMA OF THE LUNG SCIENCE Wong, A. J., Ruppert, J. M., Eggleston, J., Hamilton, S. R., BAYLIN, S. B., VOGELSTEIN, B. 1986; 233 (4762): 461-464

  Abstract

  The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.

  View details for Web of Science ID A1986D234300039

  View details for PubMedID 3014659

• DISORGANIZATION OF CULTURED VASCULAR ENDOTHELIAL CELL MONOLAYERS BY FIBRINOGEN FRAGMENT-D SCIENCE DANG, C. V., Bell, W. R., Kaiser, D., Wong, A. 1985; 227 (4693): 1487-1490

  Abstract

  Fibrinogen fragment D, which is heterogeneous, has several important biological functions. Human fibrinogen fragments D94 (molecular weight, 94,000), D78 (78,000), and E (52,000) were purified. Fragments D78 and D94 but not purified fibrinogen or fragment E specifically caused disorganization of bovine aortic endothelial cells cultured as monolayers. Within 2 hours of exposure to pathophysiological concentrations of fragment D, the confluent endothelial cells retracted from each other and projected pseudopodia. These disturbed cells subsequently became rounded and detached from the substrate. The actin present in stress fibers in stationary monolayer cells was diffusely redistributed in cells with fragment D-induced alterations in morphology. This effect was not observed in monolayers of kidney epithelial cells. The results demonstrate a specific effect of fibrinogen fragment D on the disorganization of cultured vascular endothelial cell monolayers and suggest that fragment D plays a role in the pathogenesis of syndromes with vascular endothelial damage.

  View details for Web of Science ID A1985ADG0600031

  View details for PubMedID 4038818

• MYOSIN FROM HUMAN-ERYTHROCYTES JOURNAL OF BIOLOGICAL CHEMISTRY Wong, A. J., Kiehart, D. P., Pollard, T. D. 1985; 260 (1): 46-49

  Abstract

  We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filaments like those of platelet myosin. Eight of eight monoclonal antibodies to platelet myosin also bind to erythrocyte myosin. Like most myosins, it has a high ATPase activity in the presence of Ca2+ or EDTA, but is inhibited by Mg2+. Myosin light-chain kinase transfers 1 phosphate from ATP to the 20-kDa light chain, and this stimulates the actin-activated ATPase. Thus, myosin may play a role in shape changes in the erythrocytes.

  View details for Web of Science ID A1985TZ26100003

  View details for PubMedID 3155518

• ACTIN FILAMENT STRESS FIBERS IN VASCULAR ENDOTHELIAL-CELLS INVIVO SCIENCE Wong, A. J., Pollard, T. D., Herman, I. M. 1983; 219 (4586): 867-869

  Abstract

  Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.

  View details for Web of Science ID A1983QB45400041

  View details for PubMedID 6681677

• CONTRACTILE PROTEINS IN ENDOTHELIAL-CELLS ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Herman, I. M., Pollard, T. D., Wong, A. J. 1982; 401 (DEC): 50-60

  View details for Web of Science ID A1982QD55800005

  View details for PubMedID 6963126