Plexiform neurofibroma, a benign peripheral nerve tumor, is associated with the biallelic loss of function of the NF1 tumor suppressor in Schwann cells. Here, we show that FLLL32, a small molecule inhibitor of JAK/STAT3 signaling, reduces neurofibroma growth in mice with conditional, biallelic deletion of Nf1 in the Schwann cell lineage. FLLL32 treatment or Stat3 deletion in tumor cells reduced inflammatory cytokine expression and tumor macrophage numbers in neurofibroma. Although STAT3 inhibition down-regulated the chemokines CCL2 and CCL12, which can signal through CCR2 to recruit macrophages to peripheral nerves, deletion of Ccr2 did not improve survival or reduce macrophage numbers in neurofibroma-bearing mice. Interestingly, macrophages accounted for ~20-40% of proliferating cells in untreated tumors. FLLL32 suppressed this proliferation, as well as Schwann cell proliferation, implicating STAT3-dependent, local proliferation in neurofibroma macrophage accumulation. The functions of STAT3 signaling in neurofibroma Schwann cells and macrophages, and its relevance as a therapeutic target in neurofibroma, merit further investigation.
Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Macrophages are abundant in neurofibromas, and macrophage targeted interventions may have therapeutic potential in these tumors. We generated gene expression data from fluorescence-activated cell sorted (FACS) SCs and macrophages from wild-type and mutant nerve and neurofibroma to identify candidate pathways involved in SC-macrophage cross-talk. While in 1-month-old Nf1 mutant nerve neither SCs nor macrophages significantly differed from their normal counterparts, both macrophages and SCs showed significantly altered cytokine gene expression in neurofibromas. Computationally reconstructed SC-macrophage molecular networks were enriched for inflammation-associated pathways. We verified that neurofibroma SC conditioned medium contains macrophage chemo-attractants including colony stimulation factor 1 (CSF1). Network analysis confirmed previously implicated pathways and predict novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Network analysis also predicted a central role for decreased type-I interferon signaling. We validated type-I interferon expression in neurofibroma by protein profiling, and show that treatment of neurofibroma-bearing mice with polyethylene glycolyated (PEGylated) type-I interferon-α2b reduces the expression of many cytokines overexpressed in neurofibroma. These studies reveal numerous potential targetable interactions between Nf1 mutant SCs and macrophages for further analyses.
Neurofibromatosis type 2 is an inherited, neoplastic disease associated with schwannomas, meningiomas, and ependymomas and that is caused by inactivation of the tumor suppressor gene NF2.immortalized mouse The NF2 gene product, Merlin, has no intrinsic catalytic activity; its tumor suppressor function is mediated through the proteins with which it interacts. We used proximity biotinylation followed by mass spectrometry and direct binding assays to identify proteins that associated with wild-type and various mutant forms of Merlin in immortalized Schwann cells. We defined a set of 52 proteins in close proximity to wild-type Merlin. Most of the Merlin-proximal proteins were components of cell junctional signaling complexes, suggesting that additional potential interaction partners may exist in adherens junctions, tight junctions, and focal adhesions. With mutant forms of Merlin that cannot bind to phosphatidylinositol 4,5-bisphosphate (PIP2) or that constitutively adopt a closed conformation, we confirmed a critical role for PIP2 binding in Merlin function and identified a large cohort of proteins that specifically interacted with Merlin in the closed conformation. Among these proteins, we identified a previously unreported Merlin-binding protein, apoptosis-stimulated p53 protein 2 (ASPP2, also called Tp53bp2), that bound to closed-conformation Merlin predominately through the FERM domain. Our results demonstrate that Merlin is a component of cell junctional mechanosensing complexes and defines a specific set of proteins through which it acts.
Plexiform neurofibromas (PNF) are peripheral nerve tumors caused by bi-allelic loss of NF1 in the Schwann cell (SC) lineage. PNF are common in individuals with Neurofibromatosis type I (NF1) and can cause significant patient morbidity, spurring research into potential therapies. Immune cells are rare in peripheral nerve, whereas in PNF 30% of the cells are monocytes/macrophages. Mast cells, T cells, and dendritic cells (DCs) are also present. NF1 mutant neurofibroma SCs with elevated Ras-GTP signaling resemble injury-induced repair SCs, in producing growth factors and cytokines not normally present in SCs. This provides a cytokine-rich environment facilitating PNF immune cell recruitment and fibrosis. We propose a model based on genetic and pharmacologic evidence in which, after loss of Nf1 in the SC lineage, a lag occurs. Then, mast cells and macrophages are recruited to nerve. Later, T cell/DC recruitment through CXCL10/CXCR3 drives neurofibroma initiation and sustains PNF macrophages and tumor growth. Stat3 signaling is an additional critical mediator of neurofibroma initiation, cytokine production, and PNF growth. At each stage of PNF development therapeutic benefit should be achievable through pharmacologic modulation of leukocyte recruitment and function.
The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semiquantitative analyses of changes in mRNA gene expression.
macrophage subpopulations with distinct activities (24,25). Further characterization of these neurofibroma leukocytes and their contributions to tumor initiation and growth is necessary for the development of safe and effective immunomodulatory therapies.In the Dhh-Cre Nf1 fl/fl mouse neurofibroma model, biallelic deletion of Nf1 in the Schwann cell lineage mimics the biallelic loss of NF1 in human NF1 and sporadic plexiform neurofibroma (26). The peripheral nerves of these mice appear normal at 1 month of age, but pathological changes, including mast cell and macrophage infiltration and abnormal Schwann cell proliferation, are evident at 2 months of age (26,27). By 4 months of age, these mice invariably form MRI-detectable paraspinal neurofibromas that histologically and transcriptionally resemble human plexiform neurofibroma (26,28,29). Schwann cell-specific deletion of Nf1 using other drivers can also induce nerve pathology and neurofibroma development in mice (19,(30)(31)(32). In contrast, CNPase-human EGFR (CNPase-hEGFR) mice have increased Ras activity driven by overexpression of hEGFR and develop similar nerve pathology to Nf1 mouse models but have reduced myeloid cell infiltration, and only approximately 1 in 20 develop a neurofibroma (20,33).Here, we compare nerves from these mouse models transcriptionally and identify a chemokine, Cxcl10, that is uniquely overexpressed in 2-month Dhh-Cre Nf1 fl/fl nerves. CXCL10 and its receptor, CXCR3, are important modulators of neuroinflammation and tumor biology (34)(35)(36). In this study, we identify Cxcl10and Cxcr3-expressing cell populations in nerves and neurofibroma and demonstrate the necessity of Cxcr3 for neurofibroma development in Dhh-Cre Nf1 fl/fl mice. Results Differential gene expression analyses identify Cxcl10 as a potential modulator of plexiform neurofibroma development.Peripheral nerves from GEMMs of NF1 (GEMM-NF1) (P0-CreB Nf1 fl/fl , P0-CreB Nf1 fl/-, Dhh-Cre Nf1 fl/fl ) invariably develop neurofibromas (26,31). P0-CreB and Dhh-Cre are transgenic mouse strains that express Cre recombinase in peripheral nerve Schwann cells; when used to recombine Nf1 in mice, peripheral nerves show pathological mast cell recruitment, disruption of axon and nonmyelinating Schwann cell (axon/ Remak bundle) interactions, and collagen deposition (nerve disruption). This nerve disruption phenotype precedes plexiform neurofibroma development. Although several of these changes have been proposed to contribute to neurofibroma development, similar nerve pathology is also observed in CNPase-hEGFR/ + and CNPase-hEGFR/CNPase-hEGFR mice, which recruit fewer macrophages and rarely form neurofibromas (33,37). In contrast, this pathology is not observed in Npcis mice (Nf1 +/− Trp53 +/− deletions in cis) that progress directly to malignant peripheral nerve sheath tumor (ref. 38 and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.98601DS1) or in CNPase-HRas12V mice that do not develop plexiform neurofibroma (39)....
Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Current evidence suggests that an inflammatory environment is critical for neurofibroma development and growth, however the roles of intercellular interactions between SCs and other cells in neurofibroma microenvironment are not clear yet. Neurofibromas contain numerous macrophages. We analyzed gene expression in FACS-sorted SCs and macrophages from wild type nerve and neurofibroma. Nerve macrophage transcriptomes differed from previously defined macrophage sub-populations. In 1-month-old Nf1 mutant nerve, neither SCs nor macrophages significantly differed from their counterparts in normal mice. In 7-month-old neurofibromas, macrophages showed significantly altered gene expression, as did neurofibroma SCs. Computational reconstruction of SC-macrophage molecular networks based on the gene expression data revealed extensive inflammatory-associated signaling and possible interplay between macrophages and Schwann cells. Among these were previously implicated pathways and novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Specifically, the data predicted increased cytokine expression and imbalanced type-I/type-II interferon signaling, which were confirmed by protein profiling, and normalized in neurofibroma-bearing mice treated with PEGylated interferon-α2b. These studies identify Nf1 mutant SCs interactions with macrophages, which result in chronic inflammation and neurofibroma, and provide a system in which to study early changes in benign tumorigenesis. Citation Format: Kwangmin Choi, Kakajan Komurov, Jonathan S. Fletcher, Edwin Jousma, Jose A. Cancelas, Jianqiang Wu, Nancy Ratner. An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A04.
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