Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. EGFR mutations (EGFRvIII) and PI3K hyperactivation are common in GBM, promoting tumor growth and survival, including through SREBP-1-dependent-lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. Here, studies in GBM cell lines, xenograft models and GBM clinical samples, including from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the LDL receptor. Targeting LDLR with the Liver X Receptor (LXR) agonist GW3965 caused IDOL (Inducible Degrader Of LDLR)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results demonstrate that EGFRvIII can promote tumor survival through PI3K-SREBP-1 dependent up-regulation of LDLR, and suggest a role for LXR agonists in the treatment of GBM patients.

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Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFRinhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element-binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1 (mTORC1), possibly explaining rapamycin's poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR-and EGFRvIIIdependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies. [Cancer Res 2009;69(17):6889-98]

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44high GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44high GBM but not from CD44low GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44high GBM, but not in CD44low GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKTpathway.

Commensal bacteria have emerged as an important disease factor in human Crohn's disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species. Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene. CD4؉ T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885. pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for pfiT and P. fluorescens in the pathogenesis of Crohn's disease.Human inflammatory bowel disease (IBD) represents a set of a chronic, relapsing, and remitting intestinal inflammatory disorders involving T-cell-mediated mucosal and mural destruction, with polygenic disease susceptibility (4, 13, 38). Although the etiology of these diseases remains uncertain, several lines of evidence implicate commensal bacteria as an important pathogenic element in clinical disease, particularly in Crohn's disease (CD). Patients with CD are sensitive to alteration of fecal flow, and some investigators have reported evidence of a clinical responsiveness to antibiotic therapy (21,24,34). In both pharmacologic and genetic animal models of IBD, development of disease is strictly dependent on the presence of enteric microflora (19,29,(35)(36)(37)40).The identity of colitigenic bacterial species remains uncertain. In humans, a variety of bacterial and viral species have been implicated in CD, mainly on the basis of seroreactivity (3,5,8,28,44). However, CD patients typically express elevated levels of antibodies to many bacterial proteins, perhaps due to mucosal disruption and local immunologic challenge by diverse intestinal microflora. Mycobacterium has received attention as a candidate human pathogen, based on serologic evidence, but the presence of mycobacterial DNA in lesions is controversial (9,12,31,32,43,47). Bacteroides vulgatus and Helicobacter hepaticus have been evaluated with various rodent IBD models, but other intestinal commensal organisms yet undefined are likely to account for the major colitigenic species (14,25,35,36).Our laboratory recently introduced subtractive cloning as a fresh approach to identify candidate organisms in human CD (10, 45). A novel microbial gene, I2, was isolated from lesional versus adjacent uninvolved colon tissues of a patient with CD. In a population-based study (45), the I2 gene was selectively detected in lesional CD colonic tissue,...

Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffinembedded (FFPE) clinical samples is a major challenge. Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization. Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00). Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.

Since enteric microbial composition is a distinctive and stable individual trait, microbial heterogeneity may confer lifelong, non-genetic differences between individuals. Here we report that C57BL/6 mice bearing restricted flora microbiota, a distinct but diverse resident enteric microbial community, are numerically and functionally deficient in marginal zone (MZ) B cells. Surprisingly, MZ B-cell levels are minimally affected by germ-free conditions or null mutations of various TLR signaling molecules. In contrast, MZ B-cell depletion is exquisitely dependent on cytolytic CD81 T cells, and includes targeting of a cross-reactive microbial/endogenous MHC class 1B antigen. Thus, members of certain enteric microbial communities link with CD8 1 T cells as a previously unappreciated mechanism that shapes innate immunity dependent on innate-like B cells.Key words: Animal models . B-cell development . CD8 T cells . Enteric microbiota Supporting Information available online IntroductionThe enteric microbial community is astonishingly abundant and diverse in composition [1,2]. Acquired during infancy, typically of maternal origin, its composition becomes a unique but stable trait of each individual throughout life [3]. Due to its abundance, diversity, and unculturability, there is much to learn about the functionalities and impact of the enteric microbial community on host biology. Since its composition is a distinguishing trait of each individual, it represents a potentially important epigenetic factor that may shape each individual's physiology and disease susceptibility [4]. The immune system is predicted to be a key target of these epigenetic forces, since it is highly specialized both for microbial sensing and for alterations in these signals.The innate-like and adaptive arms of the immune system have emerged as a framework for the cellular processes of Eur. J. Immunol. 2008. 38: 3411-3425 DOI 10.1002 Immunomodulation 3411 microbial defense and immunoregulation [5], recently illustrated by the reciprocal roles of dendritic cells in the composition of the enteric microbiota and its contribution to colitis susceptibility [6]. Innate-like functions are associated with the MZ and B-1 B-cell subsets [7,8], which emerge from post-medullary stages of B-cell differentiation. Although conventional follicular mature (FM) B cells function in adaptive B-lymphocyte responses, marginal zone (MZ) and B-1a B cells function in early T-independent (TI) IgM antibody responses, display elevated responsiveness through TLR, and have a limited capacity for immunologic memory [9]. The present study addresses the role of TLR responsiveness, and other modes of enteric microbial sensing, on the levels of MZ B cells in the periphery. We report three observations on the role of endogenous microbial flora in the fate of this B-cell population. First, we demonstrate that mice colonized with distinct communities of resident bacteria exhibit striking defects in innate-like B-cell populations in the spleen and body cavities respectively). Second, ...