Jon P. Williams

Large-Scale Molecular Comparison of Human Schwann Cells to Malignant Peripheral Nerve Sheath Tumor Cell Lines and Tissues

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.

Plexiform and Dermal Neurofibromas and Pigmentation Are Caused by Nf1 Loss in Desert Hedgehog-Expressing Cells

Neurofibromatosis type 1 (Nf1) mutation predisposes to benign peripheral nerve (glial) tumors called neurofibromas. The point(s) in development when Nf1 loss promotes neurofibroma formation are unknown. We show that inactivation of Nf1 in the glial lineage in vitro at embryonic day 12.5 + 1, but not earlier (neural crest) or later (mature Schwann cell), results in colony-forming cells capable of multilineage differentiation. In vivo, inactivation of Nf1 using a DhhCre driver beginning at E12.5 elicits plexiform neurofibromas, dermal neurofibromas, and pigmentation. Tumor Schwann cells uniquely show biallelic Nf1 inactivation. Peripheral nerve and tumors contain transiently proliferating Schwann cells that lose axonal contact, providing insight into early neurofibroma formation. We suggest that timing of Nf1 mutation is critical for neurofibroma formation.

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. Methodology/Principal Findings Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. Conclusions/Significance These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

Nf1 mutation expands an EGFR-dependent peripheral nerve progenitor that confers neurofibroma tumorigenic potential.

Defining growth factor requirements for progenitors facilitates their characterization and amplification. We characterize a peripheral nervous system embryonic dorsal root ganglion progenitor population using in vitro clonal sphere-formation assays. Cells differentiate into glial cells, smooth muscle/fibroblast (SM/Fb)-like cells, and neurons. Genetic and pharmacologic tools revealed that sphere formation requires signaling from the EGFR tyrosine kinase. Nf1 loss of function amplifies this progenitor pool, which becomes hypersensitive to growth factors and confers tumorigenesis. DhhCre;Nf1(fl/fl) mouse neurofibromas contain a progenitor population with similar growth requirements, potential, and marker expression. In humans, NF1 mutation predisposes to benign neurofibromas, incurable peripheral nerve tumors. Prospective identification of human EGFR(+);P75(+) neurofibroma cells enriched EGF-dependent sphere-forming cells. Neurofibroma spheres contain glial-like progenitors that differentiate into neurons and SM/Fb-like cells in vitro and form benign neurofibroma-like lesions in nude mice. We suggest that expansion of an EGFR-expressing early glial progenitor contributes to neurofibroma formation.

Mast cells can contribute to axon-glial dissociation and fibrosis in peripheral nerve

Expression of the human epidermal growth factor receptor (EGFR) in murine Schwann cells results in loss of axon-Schwann cell interactions and collagen deposition, modeling peripheral nerve response to injury and tumorigenesis. Mast cells infiltrate nerves in all three situations. We show that mast cells are present in normal mouse peripheral nerve beginning at 4 weeks of age, and that the number of mast-cells in EGFR(+) nerves increases abruptly at 5-6 weeks of age as axons and Schwann cells dissociate. The increase in mast cell number is preceded and accompanied by elevated levels of mRNAs encoding the mast-cell chemoattractants Rantes, SCF and VEGF. Genetic ablation of mast cells and bone marrow reconstitution in W(41) x EGFR(+) mice indicate a role for mast cells in loss of axon-Schwann cell interactions and collagen deposition. Pharmacological stabilization of mast cells by disodium cromoglycate administration to EGFR(+) mice also diminished loss of axon-Schwann cell interaction. Together these three lines of evidence support the hypothesis that mast cells can contribute to alterations in peripheral nerves.

Schwann cell preparation from single mouse embryos: analyses of neurofibromin function in Schwann cells.

The study of peripheral nerve function in development and disease can be facilitated by the availability of cultured cells that faithfully mimic in vivo Schwann cell growth, maturation, and differentiation. We have developed a method to establish purified mouse Schwann cell culture from a single embryo at embryonic day 12.5 (E12.5) to define the abnormalities in Schwann cells caused by loss of the neurofibromatosis type 1 (Nf1) tumor suppressor protein, the RAS-GAP neurofibromin. Our method generates 2-3 x 10(6) cells/embryo highly purified (>99.5%) mouse Schwann cells in less than 2 weeks from a single E12.5 mouse embryo. Manipulation of cell medium allows purification of a Schwann-like cell population, termed Nf1-/-TXF, that resembles a tumorigenic cell in that it grows dissociated from axons and grows rapidly, yet retains expression of Schwann cell markers. We describe the preparation and characterization of both cell types.

Cancer Screening by Systemic Administration of a Gene Delivery Vector Encoding Tumor-Selective Secretable Biomarker Expression

Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a “theragnostic” agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world.

Nf1 mutation expands an EGFR-dependent peripheral nerve progenitor population and confers tumorigenic potential

Jon P. Williams1,4, Jianqiang Wu1,4, Gunnar Johansson1, Tilat A. Rizvi1, Shyra C. Miller1, Hartmut Geiger1, Punam Malik1, Wenling Li2, Yoh-suke Mukouyama2, Jose A. Cancelas1,3, and Nancy Ratner1,* 1Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Research Foundation, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229, USA 2Laboratory of Developmental Biology, Genetics, and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA 3Hoxworth Blood Center, College of Medicine, University of Cincinnati, Cincinnati, OH 45229-7013, USA

EGFR signalling identifies an embryonic peripheral nerve progenitor cell amplified by mutation in the Nf1 gene

tumor suppressor protein encoded by NF1 has rasGap activity thus, implicating constitutive activation of the ras pathway as a major consequence of NF1 loss of function. Patients with NF1 also have increased incidence of glioblastoma formation. We have sought to model these tumors for which no effective therapies have been developed. The prognosis remains unchanged over the past three decades. According to the WHO classification, four grades of astrocytoma exist. Grade 1 is benign and also referred to as pilocytic astrocytoma. Grade II or low grade astrocytoma is characterized by infiltrative cells that home on neuronal bodies (perineural satellosis). Grade III or anaplastic astrocytoma is cell dense and highly proliferative. Grade IV or glioblastoma multiforme is characterized by pseudopalisading, necrotic foci, and intense microvascularization. All forms of astrocytoma express primitive cell markers such as nestin. In addition, all forms of astrocytoma appear throughout the brain but do not leave the CNS. These observations have led to the suggestion that the CNS provides a niche that is required for tumor growth and that spontaneous tumorigenesis occurs throughout. Historically, the prevalent model for astrocytoma formation invoked mechanisms of dedifferentiation of glial cells, followed by genetic and epigenetic signals that drive neoplastic transformation. The more recent appreciation of the existence of stem cells in the lateral ventricles and dentate gyrus has raised the question of a potential role for stem cells in tumor formation. We have undertaken to model NF1 in mice through conventional knock out and conditional knock out technologies and have developed tumor suppressor based mouse models of glioblastoma. The resultant tumors histologically and molecularly resemble human glioblastoma, including progression from Grades II through IV. Through analysis of a variety of criteria, our data have provided evidence that stem cells could account for the origin of glioblastoma. Through the use of virally transduced cre delivery to stem cell compartments as well as use of tamoxifen inducible stem cell specific cre transgenes, we have developed direct evidence for precursor cells as the origin of tumor cell.

An amplified EGF-dependent peripheral nervous system progenitor in neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) predisposes to benign, incurable peripheral nerve tumors. We hypothesized that expansion of a progenitor population in Nf1 mutant peripheral nerves contributes to tumorigenesis. To test this hypothesis, we studied progenitor cells from Nf1 mutant mice. We identified an embryonic dorsal root ganglia (DRG) derived cell population, which is expandable as EGF-dependent self-renewing spheres; loss of Nf1 is associated with an increase in sphere formation. Spheres from both wild type and Nf1 mutant DRG contained cells capable of glial differentiation. Spheres from Nf1 mutants contained cells with increased propensity to form neurons in vitro when compared with wild type cells. An EGFR+;p75+ cell population was previously isolated from Nf1 DRG cultures; here we show that these cells (Nf1−/−TXF) can be propagated as spheres at greatly (20×) increased efficiency. Consistent with a progenitor phenotype, Nf1−/−TXF cells showed migratory characteristics of neural crest stem cells in a chicken xenograft model and expressed markers (as assessed using Affymetrix GeneChips) of neural crest and at least three crest derivatives: neurons, Schwann cells, and melanocytes. Knockout of Nf1 at the embryonic Schwann cell stage of development, but not earlier or later, generated this progenitor like population, implying that loss of Nf1 at a critical stage in development causes an expansion of progenitor cells. Nf1 but not wild type perinatal mouse nerves contained cells expressing EGFR and p75, suggesting abnormal persistence of the progenitor population. We also prospectively identified EGFR+;p75+ cells by FACS analysis of human neurofibromas, and have derived spheres from primary neurofibroma cells in the presence of EGF. These data support the hypothesis that an EGFR+ neural progenitor is amplified in Nf1 mutants and is relevant to peripheral nerve tumorigenesis in NF1.