Robert G. Tuskan

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.

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.

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.

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. Mol Cancer Ther; 9(5); 1234–43. ©2010 AACR.

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.

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.

Abstract 354: PI-103 and TCN as proliferative inhibitors of the RTK/PI3K/ATK signaling pathways in astrocytomas

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC INTRODUCTION: Glioblastomas (GBMs), the most aggressive of malignant astrocytomas, are recurrent, infiltrative, and fatal. In GBMs, receptor tyrosine kinases (RTKs) are often mutated and activated, leading to the dysregulation of normal cell signaling pathways. The PI3K/Akt1/mTOR pathway, which is important in normal biological events such as proliferation, survival, invasion, migration and angiogenesis, is often deregulated in malignant cancers, resulting from the simultaneous loss of tumor suppressor PTEN, and the hyperactivation of the Akt pathway. Therefore, pharmacological inhibition of the PI3K/Akt1/mTOR may prove beneficial in arresting the proliferation of astrocytomas and glioblastomas. METHODS: In order to determine if the selected inhibitors PIA-6, [OSU03012][1], Rapamycin, Tricribine (TCN), and PI-103 had an effect on the PI3K/Akt-1/mTOR pathway, we examined their inhibitory effects on mouse astrocytoma cell lines K1861-10 Grade II; KR158 Grade III; K130G#3 Grade IV; human astrocytoma cell lines, U87MG and SF295, both Grades IV; and normal proliferating mouse primary astrocytes using the Alamar blue assay as a measure of metabolic activity and proliferation. Inhibitors with low IC50 values in tumor cells as compared to normal primary astrocytes were favored. RESULTS: From the select panel of inhibitors, only the AKT inhibitor TCN and dual PI3K/mTor inhibitor PI-103 showed low IC50 values in the nM or pM range, suggesting strong inhibition of proliferation in the astrocytoma cell lines tested. Rapamycin, an mTOR inhibitor, showed inconsistent inhibition of cell proliferation on the cell lines tested, although mTor activity was effectively inhibited, suggesting that inhibitory effects of rapamycin at higher concentrations may be acting via different pathways. CONCLUSIONS: Our results support the hypothesis that the PI3K/Akt1/mTOR pathway is a potential druggable target in astrocytomas and glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 354. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=OSU03012&atom=%2Fcanres%2F70%2F8_Supplement%2F354.atom

Abstract C12: EGFR signaling controls the chromatin binding status of BMI1 through a CK2/PP1 phosphorylation/dephosphorylation pathway and affects astrocytoma proliferation and neural stem cell differentiation

Studies of brain tumors demonstrate the importance of a rare tumor cell population with the capability of regenerating tumors upon transplantation. These cancer stem cells (CSC) are believed to be more resistant to therapy than the bulk of brain tumor cells. Studies in mouse brain tumor models demonstrate that neural stem cells (NSC) can be an important initiating cell type for brain tumors, but the relationship between NSC and CSC is unclear. EGF is a critical growth factor for NSC maintenance in culture and EGFR signaling is upregulated in glioblastoma. Similarly, the chromatin modulator BMI1 is necessary for NSC maintenance, and has been implicated in glioblastoma. We present studies linking cell surface EGF signaling and nuclear BMI1 chromatin binding. We demonstrate that BMI1 is specifically phosphorylated by EGFR signaling using EGF and TGFα, but not by other ligands tested. We have identified three amino acids encompassing the phosphorylation site in response to EGFR signaling, and show that CK2 and PP1 are necessary for control of BMI1 phosphorylation. This phosphorylation alters the chromatin binding state of BMI1 and transcriptional regulation in astrocytoma cells. Interestingly we find that BMI1 can alter transcriptional profiles in both EGF‐dependent and independent ways, suggesting that different signaling pathways could have specific effects on changing the BMI1‐dependent epigenetic state of subsets of genes. Furthermore, EGF‐induced phosphorylation of BMI1 is important for astrocytoma growth in vitro and in vivo , as well as maintenance of NSC. Our data link signals outside the cell to the nucleus important for NSC maintenance and astrocytoma proliferation. These data represent a common pathway in NSC and CSC that may explain the importance of EGF signaling for NSC maintenance and gliomagenesis, and suggest a novel downstream pathway (CK2/PP1) involved in these processes. Citation Information: Cancer Res 2009;69(23 Suppl):C12.