Control of proliferation in astrocytoma cells by the receptor tyrosine kinase/PI3K/AKT signaling axis and the use of PI-103 and TCN as potential anti-astrocytoma therapies.

A growing body of work suggests that astrocytomas and glioblastoma multiforme will require carefully tailored, molecularly targeted therapy for successful treatment. Recent efforts to comprehensively identify mutations and gene expression changes in glioblastoma have shown that mutation of NF1 is a common alteration in human glioblastoma. We have developed and characterized a panel of 14 tumor lines from grades II through IV astrocytomas developed from our Nf1-/+;Trp53-/+cis mouse model and have used this panel to characterize signal transduction pathways and inhibitors that are candidate therapeutic targets for astrocytoma and glioblastoma. We show that these tumors express platelet-derived growth factor receptor-α, epidermal growth factor receptor, and their respective ligands to varying degrees. We find that both the MEK and PI3K signaling pathways downstream of epidermal growth factor receptor and platelet-derived growth factor receptor-α are necessary for full proliferation of astrocytoma cells; however, inhibition of the PI3K pathway is more effective than inhibition of MEK at blocking cell growth. We have examined inhibitors of the PI3K/Akt/mTOR signaling pathway and find that PI-103 and TCN show particular promise for inhibiting growth in Nf1 and Trp53 mutant astrocytoma cells.

Schweinfurthin A selectively inhibits proliferation and Rho signaling in glioma and neurofibromatosis type 1 tumor cells in a NF1-GRD-dependent manner.

Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1-/+;Trp53-/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1-/-;Trp53-/- astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor-stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling.

The neurofibromatosis type 1 tumor suppressor controls cell growth by regulating signal transducer and activator of transcription-3 activity in vitro and in vivo.

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome in which affected individuals develop benign and malignant nerve tumors. The NF1 gene product neurofibromin negatively regulates Ras and mammalian target of rapamycin (mTOR) signaling, prompting clinical trials to evaluate the ability of Ras and mTOR pathway inhibitors to arrest NF1-associated tumor growth. To discover other downstream targets of neurofibromin, we performed an unbiased cell-based high-throughput chemical library screen using NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells. We identified the natural product, cucurbitacin-I (JSI-124), which inhibited NF1-deficient cell growth by inducing apoptosis. We further showed that signal transducer and activator of transcription-3 (STAT3), the target of cucurbitacin-I inhibition, was hyperactivated in NF1-deficient primary astrocytes and neural stem cells, mouse glioma cells, and human MPNST cells through Ser(727) phosphorylation, leading to increased cyclin D1 expression. STAT3 was regulated in NF1-deficient cells of murine and human origin in a TORC1- and Rac1-dependent manner. Finally, cucurbitacin-I inhibited the growth of NF1-deficient MPNST cells in vivo. In summary, we used a chemical genetics approach to reveal STAT3 as a novel neurofibromin/mTOR pathway signaling molecule, define its action and regulation, and establish STAT3 as a tractable target for future NF1-associated cancer therapy studies.

Real-time PCR analysis of candidate imprinted genes on mouse chromosome 11 shows balanced expression from the maternal and paternal chromosomes and strain-specific variation in expression levels.

Imprinted genes are monoallelically expressed from either the maternal or paternal genome. Because cancer develops through genetic and epigenetic alterations, imprinted genes affect tumorigenesis depending on which parental allele undergoes alteration. We have shown previously in a mouse model of neurofibromatosis type 1 (NF1) that inheriting mutant alleles of Nf1 and Trp53 on chromosome 11 from the mother or father dramatically changes the tumor spectrum of mutant progeny, likely due to alteration in an imprinted gene(s) linked to Nf1 and Trp53. In order to identify imprinted genes on chromosome 11 that are responsible for differences in susceptibility, we tested candidate imprinted genes predicted by a bioinformatics approach and an experimental approach. We have tested 30 candidate genes (Havcr2, Camk2b, Ccdc85a, Cntnap1, Ikzf1, 5730522E02Rik, Gria1, Zfp39, Sgcd, Jup, Nxph3, Spnb2, Asb3, Rasd1, Map2k3, Map2k4, Trp53, Serpinf1, Crk, Rasl10b, Itga3, Hoxb5, Cbx1, Pparbp, Igfbp4, Smarce1, Stat3, Atp6v0a1, Nbr1 and Meox1), two known imprinted genes (Grb10 and Impact) and Nf1, which has not been previously identified as an imprinted gene. Although we confirmed the imprinting of Grb10 and Impact, we found no other genes imprinted in the brain. We did, however, find strain-biased expression of Camk2b, 5730522E02Rik, Havcr2, Map2k3, Serpinf1, Rasl10b, Itga3, Asb3, Trp53, Nf1, Smarce1, Stat3, Cbx1, Pparbp and Cntnap1. These results suggest that the prediction of imprinted genes is complicated and must be individually validated. This manuscript includes supplementary data listing primer sequences for Taqman assays and Ct values for Taqman PCR.

Nf1 expression is dependent on strain background: implications for tumor suppressor haploinsufficiency studies.

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.

An imprinted locus epistatically influences Nstr1 and Nstr2 to control resistance to nerve sheath tumors in a neurofibromatosis type 1 mouse model.

Cancer is a complex disease in which cells acquire many genetic and epigenetic alterations. We have examined how three types of alterations, mutations in tumor suppressor genes, changes in an imprinted locus, and polymorphic loci, interact to affect tumor susceptibility in a mouse model of neurofibromatosis type 1 (NF1). Mutations in tumor suppressor genes such as TP53 and in oncogenes such as KRAS have major effects on tumorigenesis due to the central roles of these genes in cell proliferation and cell survival. Imprinted genes expressed from only one parental chromosome affect tumorigenesis if their monoallelic expression is lost or duplicated. Because imprinted loci are within regions deleted or amplified in cancer, the parental origin of genomic rearrangements could affect tumorigenesis. Gene polymorphisms can vary tumor incidence by affecting rate-limiting steps in tumorigenesis within tumor cells or surrounding stroma. In our mouse model of NF1, the incidence of tumors mutant for the tumor suppressor genes Nf1 and Trp53 is strongly modified by a linked imprinted locus acting epistatically on two unlinked polymorphic loci, Nstr1 and Nstr2. This interaction of an imprinted locus and polymorphic susceptibility loci has profound implications for human mapping studies where the parental contribution of alleles is often unknown.

Cholera toxin indirectly activates human monocyte-derived dendritic cells in vitro through the production of soluble factors, including prostaglandin E(2) and nitric oxide.

Cholera toxin (CT) is a potent adjuvant that activates dendritic cells (DC) by increasing intracellular cyclic AMP (cAMP) levels. In vivo and in vitro, very small amounts of CT induce potent adjuvant effects and activate DC. We hypothesized that DC intoxicated by CT may release factors that enhance their own maturation and induce the maturation of toxin-free bystander DC. Through the use of mixed cultures and transwell cultures, we found that human monocyte-derived DC (MDDC) pulsed with CT or other cAMP-elevating agonists induce the maturation of bystander DC. Many DC agonists including CT increase the production of prostaglandin E(2) (PGE(2)) and nitric oxide (NO). For this reason, we determined whether the actions of PGE(2) or NO are involved in the maturation of MDDC induced by CT or dibutyryl-cAMP (d-cAMP). We found that blocking the production of PGE(2) or blocking prostaglandin receptors inhibited MDDC maturation induced by CT and d-cAMP. Likewise, sequestering NO or blocking the downstream actions of NO resulted in the inhibition of MDDC maturation induced by CT and d-cAMP. These results indicate that endogenously produced factors including PGE(2) and NO contribute to the maturation of DC induced by CT and that these factors participate in bystander DC maturation. The results of this study may help explain why bacterial toxins that elevate cAMP are such potent adjuvants.

Pasteurella multocida toxin activates human monocyte-derived and murine bone marrow-derived dendritic cells in vitro but suppresses antibody production in vivo.

Pasteurella multocida toxin (PMT) is a potent mitogen for fibroblasts and osteoblastic cells. PMT activates phospholipase C-beta through G(q)alpha, and the activation of this pathway is responsible for its mitogenic activity. Here, we investigated the effects of PMT on human monocyte-derived dendritic cells (MDDC) in vitro and show a novel activity for PMT. In this regard, PMT activates MDDC to mature in a dose-dependent manner through the activation of phospholipase C and subsequent mobilization of calcium. This activation was accompanied by enhanced stimulation of naive alloreactive T cells and dominant inhibition of interleukin-12 production in the presence of saturating concentrations of lipopolysaccharide. Surprisingly, although PMT mimics the activating effects of cholera toxin on human MDDC and mouse bone marrow-derived dendritic cells, we found that PMT is not a mucosal adjuvant and that it suppresses the adjuvant effects of cholera toxin in mice. Together, these results indicate discordant effects for PMT in vitro compared to those in vivo.

Susceptibility to astrocytoma in mice mutant for Nf1 and Trp53 is linked to chromosome 11 and subject to epigenetic effects.

Astrocytoma is the most common malignant brain tumor in humans. Loss of the p53 signaling pathway and up-regulation of the ras signaling pathway are common during tumor progression. We have shown previously that mice mutant for Trp53 and Nf1 develop astrocytoma, progressing to glioblastoma, on a C57BL/6J strain background. In contrast, here we present data that mice mutant for Trp53 and Nf1 on a 129S4/SvJae background are highly resistant to developing astrocytoma. Through analysis of F1 progeny, we demonstrate that susceptibility to astrocytoma is linked to chromosome 11, and that the modifier gene(s) responsible for differences in susceptibility is closely linked to Nf1 and Trp53. Furthermore, this modifier of astrocytoma susceptibility is itself epigenetically modified. These data demonstrate that epigenetic effects can have a strong effect on whether cancer develops in the context of mutant ras signaling and mutant p53, and that this mouse model of astrocytoma can be used to identify modifier phenotypes with complex inheritance patterns that would be unidentifiable in humans. Because analysis of gene function in the mouse is often performed on a mixed C57BL/6,129 strain background, these data also provide a powerful example of the potential of these strains to mask interesting gene functions.

Calcium signaling through phospholipase C activates dendritic cells to mature and is necessary for the activation and maturation of dendritic cells induced by diverse agonists.

Calcium is an important second messenger in the phospholipase C (PLC) signal transduction pathway. Calcium signaling is involved in many biological processes, including muscle contraction, cellular activation, and cellular proliferation. Dendritic cell (DC) maturation is induced by many different stimuli, including bacterial lipopolysaccharide (LPS), bacterial toxins, inflammatory cytokines, prostaglandins, as well as calcium mobilization. In the present study, we determined the role of the PLC signal transduction pathway in the activation and maturation of human monocyte-derived DCs (MDDCs) induced by diverse agonists. We found that signaling through PLC activates MDDCs to mature and is necessary for LPS, cholera toxin, dibutyryl-cyclic AMP, prostaglandin E2, and the calcium ionophore A23187 to induce MDDC maturation. The results of the present study along with the results of other studies indicate that multiple signaling pathways are involved in the activation of DCs and that inhibition of any of these pathways inhibits the maturation of DCs.

An enzymatically active a domain is required for cholera-like enterotoxins to induce a long-lived blockade on the induction of oral tolerance: new method for screening mucosal adjuvants.

The cholera-like enterotoxins (CLETS), cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), are powerful mucosal adjuvants. Here we show that these toxins also induce a long-lived blockade (of at least 6 months) on the induction of oral tolerance when they are coadministered with the antigen ovalbumin. Strikingly, only enzymatically active CLETS induced this blockade on the induction of oral tolerance. In this regard, the enzymatically inactive mutants of CT and LT, CTK63 and LTK63, and their recombinant B pentamers, rCTB and rLTB, failed to block the induction of oral tolerance, demonstrating a stringent requirement for an enzymatically active A domain in this phenomenon. Together with the results of other recent studies, these results indicate that the enzymatic activity of CLETS, most likely cyclic AMP elevation, is responsible for their adjuvant effects. The results of this study also indicate that measuring the ability of putative mucosal adjuvants to block the induction of oral tolerance may be a superior method for measuring mucosal adjuvanticity.

Pertussis toxin and the adenylate cyclase toxin from Bordetella pertussis activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a cAMP-dependent pathway.

Pertussis toxin (PT) and adenylate cyclase toxin (AT) are AB enterotoxins produced by Bordetella pertussis. PT is a powerful mucosal adjuvant whose cellular target and mechanism of action are unknown; however, emerging evidence suggests that dendritic cells (DC) may be a principal adjuvant target of PT. Here, we investigate the mechanism underlying the effects of these toxins on human monocyte-derived DC (MDDC) in vitro. We found that the effects of PT and AT on MDDC, including maturation, are mediated by cyclic adenosine monophosphate (cAMP). In this regard, adenosine 5'-diphosphate-ribosylation-defective derivatives of PT failed to induce maturation of MDDC, whereas dibutyryl-cAMP (d-cAMP) and Forskolin mimic the maturation of MDDC and dominant inhibition of cytokine production induced by these toxins. Also, cAMP-dependent kinase inhibitors blocked the ability of PT, AT, d-cAMP, and Forskolin to activate MDDC. Taken together, these results show that the effects of PT and AT on MDDC are mediated strictly by cAMP.

Cholera toxin and heat-labile enterotoxin activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a cyclic AMP-dependent pathway.

Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown. There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo. Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro. We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent. ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3',5'-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT. In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC. CT, LT, dibutyryl-cyclic-3',5'-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide. Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.