IGFBP2 potentiates nuclear EGFR-STAT3 signaling.

Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of epidermal growth factor receptor (EGFR), which subsequently activates signal transducer and activator of transcription factor 3 (STAT3) signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from The Cancer Genome Atlas database for human glioma. A high level of all three proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient data set. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by two distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target.

Neutralizing the EGF receptor in glioblastoma cells stimulates cell migration by activating uPAR-initiated cell signaling.

In glioblastoma (GBM), the EGF receptor (EGFR) and Src family kinases (SFKs) contribute to an aggressive phenotype. EGFR may be targeted therapeutically; however, resistance to EGFR-targeting drugs such as Erlotinib and Gefitinib develops quickly. In many GBMs, a truncated form of the EGFR (EGFRvIII) is expressed. Although EGFRvIII is constitutively active and promotes cancer progression, its activity is attenuated compared with EGF-ligated wild-type EGFR, suggesting that EGFRvIII may function together with other signaling receptors in cancer cells to induce an aggressive phenotype. In this study, we demonstrate that in EGFRvIII-expressing GBM cells, the urokinase receptor (uPAR) functions as a major activator of SFKs, controlling phosphorylation of downstream targets, such as p130Cas and Tyr-845 in the EGFR in vitro and in vivo. When EGFRvIII expression in GBM cells was neutralized, either genetically or by treating the cells with Gefitinib, paradoxically, the cells demonstrated increased cell migration. The increase in cell migration was explained by a compensatory increase in expression of urokinase-type plasminogen activator, which activates uPAR-dependent cell signaling. GBM cells that were selected for their ability to grow in vivo in the absence of EGFRvIII also demonstrated increased cell migration, due to activation of the uPAR signaling system. The increase in GBM cell migration, induced by genetic or pharmacologic targeting of the EGFR, was blocked by Dasatinib, highlighting the central role of SFKs in uPAR-promoted cell migration. These results suggest that compensatory activation of uPAR-dependent cell signaling, in GBM cells treated with targeted therapeutics, may adversely affect the course of the disease by promoting cell migration, which may be associated with tumor progression.

EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180.

Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H-89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180(S1250L) mutant, but not wild type Dock180(WT), protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to develop potential therapeutic strategies for malignant gliomas.

Targeting EGFR for treatment of glioblastoma: molecular basis to overcome resistance.

Glioblastoma (glioblastoma multiforme; GBM; WHO Grade IV) accounts for the majority of primary malignant brain tumors in adults. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene represent signature genetic abnormalities encountered in GBM. A range of potential therapies that target EGFR or its mutant constitutively active form, ΔEGFR, including tyrosine kinase inhibitors (TKIs), monoclonal antibodies, vaccines, and RNA-based agents, are currently in development or in clinical trials for the treatment of GBM. Data from experimental studies evaluating these therapies have been very promising; however, their efficacy in the clinic has so far been limited by both upfront and acquired drug resistance. This review discusses the current status of anti-EGFR agents and the recurrent problem of resistance to these agents that strongly indicates that a multiple target approach will provide a more favorable future for these types of targeted therapies in GBM.

EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway.

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPß. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Vascular endothelial growth factor isoforms display distinct activities in promoting tumor angiogenesis at different anatomic sites.

The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(121) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing vascularization and growth of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(121) only showed enhancement of i.c. tumor growth but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neovascularization, whereas VEGF(121) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. Endothelial cell migration stimulated by VEGF(121) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.

Human glioblastoma xenografts overexpressing a tumor-specific mutant epidermal growth factor receptor sensitized to cisplatin by the AG1478 tyrosine kinase inhibitor.

OBJECT: Activation of signaling by the epidermal growth factor receptor (EGFR) through gene amplification or rearrangement is common in human malignancy, especially in a large fraction of de novo glioblastomas multiforme (GBMs). The most common mutant EGFR, (AEGFR, also known as de2-7 EGFR and EGFRvIII) lacks a portion of the extracellular domain, enhances tumorigenicity in vivo, and causes resistance to the chemotherapeutic drug cisplatin (CDDP). This resistance is due to the suppression of CDDP-induced apoptosis by the constitutively active tyrosine kinase activity of the receptor. The authors have investigated whether inhibition of AEGFR signaling by the tyrosine kinase inhibitor, tyrphostin AG1478, could sensitize tumor xenografts to CDDP and, thereby, enhance its therapeutic efficacy in animals. METHODS: Nude mice were inoculated either subcutaneously or intracerebrally with human GBM cells expressing AEGFR and were then systemically treated with CDDP and/or AG1478. Tumor volumes were monitored and tumor sections were analyzed by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays or MIB-1 staining. Expression of AEGFR, but not wild-type EGFR, conferred CDDP resistance to the cells in vivo. Inhibition of receptor signaling by the EGFR-specific tyrosine kinase inhibitor, AG1478. sensitized the xenografts to the cytotoxic effects of CDDP. This combined CDDP/AG1478 treatment significantly suppressed growth of subcutaneous xenografts in nude mice in a synergistic manner (p < 0.01 compared with vehicle control) without causing generalized toxicity, whereas treatments with CDDP or AG1478 alone were ineffective. The synergistic growth suppression by the CDDP/AG1478 combination was not observed in xenografts overexpressing wild-type EGFR or kinase-deficient AEGFR. The combined CDDP/ AG1478 treatment induced tumor growth suppression, which correlated with increased apoptosis and reduced proliferation. This treatment also extended the life span of mice bearing intracerebral xenografts (p < 0.01 compared with controls). CONCLUSIONS: The results of this study may provide the basis for the development of a novel and safe therapeutic strategy for the very aggressive AEGFR-expressing GBM.

Trypanosoma cruzi trans-sialidase: a potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling.

Patients infected with Trypanosoma cruzi may remain asymptomatic for decades and show signs of neuroregeneration in the peripheral nervous system (PNS). In the absence of such neuroregeneration, patients may die in part by extensive neuronal destruction in the gastrointestinal tract. Thus, T. cruzi may, despite their invasion of the PNS, directly prevent cell death to keep nerve destruction in check. Indeed, T. cruzi invasion of Schwann cells, their prime target in PNS, suppressed host-cell apoptosis caused by growth-factor deprivation. The trans-sialidase (TS) of T. cruzi and the Cys-rich domain of TS reproduced the antiapoptotic activity of the parasites at doses (> or =3.0 nM) comparable or lower than those of bona fide mammalian growth factors. This effect was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). TS also activated Akt, a downstream effector of PI3K. Ectopic expression of TS in an unrelated parasite, Leishmania major, turned those parasites into activators of Akt in Schwann cells. In contrast, the Cys-rich domain of TS did not block apoptosis in Schwann cells overexpressing dominant-negative Akt or constitutively active PTEN, a negative regulator of PI3K/Akt signaling. The results demonstrate that T. cruzi, through its TS, triggers the survival of host Schwann cells via the PI3K/Akt pathway, suggesting a role for PI3K/Akt in the pathogenesis of Chagas' disease.

The protein tyrosine phosphatase TCPTP suppresses the tumorigenicity of glioblastoma cells expressing a mutant epidermal growth factor receptor.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and GBMs frequently contain amplifications or mutations of the EGFR gene. The most common mutation results in a truncated receptor tyrosine kinase known as Delta EGFR that signals constitutively and promotes GBM growth. Here, we report that the 45-kDa variant of the protein tyrosine phosphatase TCPTP (TC45) can recognize Delta EGFR as a cellular substrate. TC45 dephosphorylated Delta EGFR in U87MG glioblastoma cells and inhibited mitogen-activated protein kinase ERK2 and phosphatidylinositol 3-kinase signaling. In contrast, the substrate-trapping TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, suppressed the activation of ERK2 but not phosphatidylinositol 3-kinase. TC45 inhibited the proliferation and anchorage-independent growth of Delta EGFR cells but TC45-D182A only inhibited cellular proliferation. Notably, neither TC45 nor TC45-D182A inhibited the proliferation of U87MG cells that did not express Delta EGFR. Delta EGFR activity was necessary for the activation of ERK2, and pharmacological inhibition of ERK2 inhibited the proliferation of Delta EGFR-expressing U87MG cells. Expression of either TC45 or TC45-D182A also suppressed the growth of Delta EGFR-expressing U87MG cells in vivo and prolonged the survival of mice implanted intracerebrally with these tumor cells. These results indicate that TC45 can inhibit the Delta EGFR-mediated activation of ERK2 and suppress the tumorigenicity of Delta EGFR-expressing glioblastoma cells in vivo.

Growth suppression of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor.

A mutant epidermal growth factor receptor (variously called DeltaEGFR, de2-7 EGFR, or EGFRvIII) containing a deletion of 267 amino acids of the extracellular domain is frequently highly expressed in human malignant gliomas and has been reported for cancers of the lung, breast, and prostate. We tested the efficacy of a novel monoclonal anti-DeltaEGFR antibody, mAb 806, on the growth of intracranial xenografted gliomas in nude mice. Systemic treatment with mAb 806 significantly reduced the volume of tumors and increased the survival of mice bearing xenografts of U87 MG.DeltaEGFR, LN-Z308.DeltaEGFR, or A1207.DeltaEGFR gliomas, each of which expresses high levels of DeltaEGFR. In contrast, mAb 806 treatment was ineffective with mice bearing the parental U87 MG tumors, which expressed low levels of endogenous wild-type EGFR, or U87 MG.DK tumors, which expressed high levels of kinase-deficient DeltaEGFR. A slight increase of survival of mice xenografted with a wild-type EGFR-overexpressing U87 MG glioma (U87 MG.wtEGFR) was effected by mAb 806 concordant with its weak cross-reactivity with such cells. Treatment of U87 MG.DeltaEGFR tumors in mice with mAb 806 caused decreases in both tumor growth and angiogenesis, as well as increased apoptosis. Mechanistically, in vivo mAb 806 treatment resulted in reduced phosphorylation of the constitutively active DeltaEGFR and caused down-regulated expression of the apoptotic protector, Bcl-XL. These data provide preclinical evidence that mAb 806 treatment may be a useful biotherapeutic agent for those aggressive gliomas that express DeltaEGFR.

Monoclonal antibody 806 inhibits the growth of tumor xenografts expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR.

The monoclonal antibody (mAb) 806 was raised against the delta2-7 epidermal growth factor receptor (de2-7 EGFR or EGFRvIII), a truncated version of the EGFR commonly expressed in glioma. Unexpectedly, mAb 806 also bound the EGFR expressed by cells exhibiting amplification of the EGFR gene but not to cells or normal tissue expressing the wild-type receptor in the absence of gene amplification. The unique specificity of mAb 806 offers an advantage over current EGFR antibodies, which all display significant binding to the liver and skin in humans. Therefore, we examined the antitumor activity of mAb 806 against human tumor xenografts grown in nude mice. The growth of U87 MG xenografts, a glioma cell line that endogenously expresses approximately 10(5) EGFRs in the absence of gene amplification, was not inhibited by mAb 806. In contrast, mAb 806 significantly inhibited the growth of U87 MG xenografts transfected with the de2-7 EGFR in a dose-dependent manner using both preventative and established tumor models. Significantly, U87 MG cells transfected with the wild-type EGFR, which increased expression to approximately 10(6) EGFRs/cell and mimics the situation of gene amplification, were also inhibited by mAb 806 when grown as xenografts in nude mice. Xenografts treated with mAb 806 all displayed large areas of necrosis that were absent in control tumors. This reduced xenograft viability was not mediated by receptor down-regulation or clonal selection because levels of antigen expression were similar in control and treated groups. The antitumor effect of mAb 806 was not restricted to U87 MG cells because the antibody inhibited the growth of new and established A431 xenografts, a cell line expressing >10(6) EGFRs/cell. This study demonstrates that mAb 806 possesses significant antitumor activity.

The potential of TRAIL for cancer chemotherapy.

Innate and acquired resistance to chemotherapy and radiation therapy has been a major obstacle for clinical oncology. One potential adjunct to such conventional treatments is direct induction of cell death by activation of death receptor-mediated apoptosis. TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), a recently identified member of the growing TNF superfamily, binds to its cognate "death" receptors DR4 and DR5 as well as "decoy" receptors DcR1 and DcR2. Upon binding, rapid apoptosis is enacted in a variety of human cancer cell lines independent of p53 status, but not in normal cell lines. TRAIL treatment results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice. Furthermore, combination treatment of TRAIL with genotoxic chemotherapeutic agents synergistically suppresses growth of tumor xenografts which are otherwise resistant to treatment with TRAIL or chemotherapy alone. Unlike the other death ligands TNF-alpha or FasL, systemic administration of soluble human TRAIL does not cause toxicity in mice and non-human primates. While further studies are needed to evaluate the possible cytotoxicity of TRAIL especially for human hepatocytes, indications are increasing that TRAIL may be a novel therapeutic agent for human cancer.

Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse.

Genetic alterations of FKHR (FOXO1), AF6q21 (FOXO2), and AFX (FOXO4), closely related members of the forkhead family of DNA binding proteins, in human cancers has suggested they play a role in the regulation of cellular differentiation or proliferation. In order to elucidate the function of this gene subfamily during mammalian development, we have identified and characterized three novel mouse genes; Fkhr1 (Foxo1), Fkhr2 (Foxo3), and Afxh (Foxo4), which are closely related to the human FKHR (Foxo1), AF6q21 (FOXO2), and AFX (FOXO4) genes, respectively. The genes are each expressed both during development and in the adult with distinct patterns ranging from ubiquitous [Fkhr2 (Foxo3)] to tissue-specific [Afxh (Foxo4)]. Selection of high-affinity DNA-binding sites from a pool of degenerate oligonucleotides demonstrated that the proteins encoded by these genes recognize a core sequence [(T/A) (A/T) A A C A] similar to that recognized by other forkhead domain-containing proteins. We have also identified additional FKHR-related genes expressed during development in both the chick and zebrafish. Further characterization will provide insight into the roles of members of the FKHR subfamily of forkhead-related genes during both normal and neoplastic development.

Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells.

The sensitivity of non-isotopic PCR-SSCP was compared to direct sequencing of PTEN exons. DNA from leukocytes derived from healthy donors, and from the glioblastoma cell line LN319 was extracted and mixed in different proportions from 0 to 100%. The LN319 cell line contains a point mutation at codon 15 exon 1 of the PTEN gene. The PCR-SSCP experiments demonstrated mutations in samples containing as little as 10% tumor DNA. In contrast, direct DNA sequencing experiments were less sensitive, requiring 30-70% of tumor DNA in the sample, depending on the DNA strand sequenced. In conclusion, PCR-SSCP, in our hands, is more sensitive than automated sequencing for detecting PTEN point mutations. We recommend to always sequence both strands, and take into account that samples containing less than 30% tumor cells should not only be sequenced, but also studied by PCR-SSCP in order to discriminate false negative results.

Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3.

A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter/enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.

Aberrant receptor signaling in human malignant gliomas: mechanisms and therapeutic implications.

Alterations of the epidermal growth factor receptor (EGFR) occur frequently in malignant gliomas through gene amplification or rearrangement, especially in a large fraction of de novo type glioblastomas. The most common of these mutant EGFRs (variously named de2-7 EGFR, deltaEGFR or EGFRvIII) lacks a portion of the extracellular ligand-binding domain. Here, we review the evidence that shows that expression of deltaEGFR bestows in vivo growth advantages to human glioma cells through its constitutively active tyrosine kinase activity. Thus, deltaEGFR may provide a novel therapeutic target for the most aggressive type of glioblastoma.

Analysis of the p300/CBP-Associated Factor (PCAF) gene in astrocytic tumors.

The PCAF gene encodes the p300/CBP-Associated Factor (PCAF), a histone acetyltransferase, which regulates p53 by acetylation of Lys320 in the C-terminal portion of p53. While the p53 gene is one of the most frequently mutated tumor suppressor genes in human tumors, such mutations occur in only 30% of astrocytic tumors. Since PCAF can regulate p53 activity, abrogation of PCAF function by PCAF gene mutation could be an alternate mechanism to inactivate the p53 pathway in tumors lacking p53 mutations. To test this hypothesis, we determined the nucleotide sequence of the entire PCAF coding region in 37 astrocytic tumors (17 glioblastomas, 10 anaplastic astrocytomas, 7 low-grade astrocytomas, and 3 pilocytic astrocytomas). We detected two single-nucleotide alterations that represented non-deleterious polymorphisms (GAG > GAA Glu103Glu, AAT > AGT Asn386Ser) but no obvious functional mutations. Moreover, the frequency of the Asn386Ser allele that contained Ser386 in glioma patients was not statistically different from its frequency in individuals without disease, and no significant association was observed between the PCAF polymorphisms and the presence or absence of p53 mutations in the tumors. We conclude that the PCAF gene is not mutated during the development of the astrocytic tumors studied here.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin.

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of A6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. A6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, A6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of A6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or A6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving A6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.