Cyclic AMP suppression is sufficient to induce gliomagenesis in a mouse model of neurofibromatosis-1

Abstract

Current models of oncogenesis incorporate the contributions of chronic inflammation and aging to the patterns of tumor formation. These oncogenic pathways, involving leukocytes and fibroblasts, are not readily applicable to brain tumors (glioma), and other mechanisms must account for microenvironmental influences on central nervous system tumorigenesis. Previous studies from our laboratories have used neurofibromatosis-1 (NF1) genetically engineered mouse (GEM) models to understand the spatial restriction of glioma formation to the optic pathway of young children. Based on our initial findings, we hypothesize that brain region-specific differences in cAMP levels account for the pattern of NF1 gliomagenesis. To provide evidence that low levels of cAMP promote glioma formation in NF1, we generated foci of decreased cAMP in brain regions where gliomas rarely form in children with NF1. Focal cAMP reduction was achieved by forced expression of phosphodiesterase 4A1 (PDE4A1) in the cortex of Nf1 GEM strains. Ectopic PDE4A1 expression produced hypercellular lesions with features of human NF1-associated glioma. Conversely, pharmacologic elevation of cAMP with the PDE4 inhibitor rolipram dramatically inhibited optic glioma growth and tumor size in Nf1 GEM in vivo. Together, these results indicate that low levels of cAMP in a susceptible Nf1 mouse strain are sufficient to promote gliomagenesis, and justify the implementation of cAMP-based stroma-targeted therapies for glioma.

Figure 1. Optic nerve gliomas in humans and mice

(A) An optic nerve glioma specimen from a patient with NF1 demonstrates mild hypercellularity and CXCL12 expression in the endothelium of tumor-associated blood vessels (e) as well as in scattered infiltrating cells. (B) Serial section from the tumor presented in panel A stained for the presence of a ligand-induced phosphorylated form of CXCR4 (pCXCR4) reveals a high level of receptor activation in proximity to the CXCL12-expressing endothelium. Phosphorylated CXCR4 is present in a pilocytic cell (inset). (C) A hypercellular lesion with nuclear atypia is evident within the optic nerve of an OPG mouse with hematoxylin and eosin stain. (D) The tumor pictured in panel C contains GFAP-expressing cells with the elongated bipolar morphology characteristic of pilocytic cells. In all cases expression appears brown. Scale bars for A and B equal 20 microns and for C and D equal 50 microns.

Figure 2. Induced tumors occur at PDE4A injection sites

Tumors were identified in eight of 13 OPG mice injected with catalytically-active PDE4A1. The following representative images were taken of four such induced tumors: (A) Induced tumors had foci of cellular clusters and nuclear atypia evident on hematoxylin and eosin stained sections. Scale bar = 25 µm. (B) Injection sites were further identified as hypercellular, disorganized lesions expressing virally encoded mCherry fluorescent protein (brown, scale bar equals 25 µm), and (C) PDE4A in characteristic perinuclear localization (yellow). DAPI counterstains nuclei blue. Scale bar = 25 µm. (D) Glial Fibrillary Acidic protein (GFAP)-expressing cells (brown) display an elongated bipolar phenotype (arrows) characteristic of pilocytic cells. Scale bar = 50 µm. Inset: Co-labelling of a piloid cell with antibodies directed against GFAP (green) and PDE4 (red) support the role of PDE4A1 expression and cAMP suppression in the genesis of glioma.

Figure 3. Induced tumors exhibit increased Olig2 expression and evidence for decreased cAMP levels

(A) Cells within induced tumors exhibited increased Olig2 expression. Scale bar = 25 μm. (B) The number of Olig2 positive nuclei per high-powered field in induced tumors was greater than in contralateral cortex. P<0.005 as determined by two-tailed t-test. (C) Immunolabeling of injection sites with antibody directed against the phosphorylated form of PKA substrates revealed a high level of staining in normal cortex (arrowhead) but low level of staining in tumor cells (asterisk). (D) Serial sections through the same tumor site pictured in C and labeled with antibody directed against mCherry fluorescent protein reveals the interface between tumor (asterisk) and cortex (arrowhead). Scale bars equal 50 microns.

Figure 4. Histological features of non-tumor reactions

In 31 cases, no induced tumor was identified in GEM brains injected with PDE4A1 or PDE4A1-H229Q. Pictured is the sequential assessment of an injection site from a single animal with positive BLI but negative tumor evaluation. (A) The injection site, which had been identified by Hematoxylin and Eosin stain and PDE4A expression (not shown), demonstrated expression of mCherry fluorescent protein. (B) There are numerous GFAP positive cells at the injection site, but they display a stellate morphology typical of reactive (nonneoplastic) astrocytes. (C) There is a lack of increased Olig2 staining. (D) Also evident at this injection site is an inflammatory response characterized by numerous LCA-positive leukocytes. In all cases expression appears brown. Scale bars for large images are 200 µm. Inset scale bars = 25 µm.

Figure 5. Targeted inhibition of PDE4 attenuates tumor growth in OPG mice

(A) Optic nerve gliomas were identified by enlarged optic nerves (white areas within the boxed region and demarcated by dotted line in inset) in OPG mice by MRI and assigned to treatment (Rolipram) or control (Vehicle) groups. (B) Optic nerve volumes in animals treated for 1 or 4 weeks with Rolipram were compared. The arrowheads indicate thickened optic nerves characteristic of these tumors. After 4 weeks of Rolipram treatment, optic nerves returned to a normal diameter. (C) Quantitation of optic nerve volumes indicates that there is a statistically significant increase in optic nerve volumes in untreated mice compared to wildtype mice (WT, without tumors) and that Rolipram abrogates this difference in volume (n = 5 mice/group). (D) The anti-tumor effect of Rolipram was associated with inhibition of tumor cell proliferation as determined by Ki67 staining and the numbers of Ki67 positive cells per high power field (HPF). N = 5 mice/group. * = p<0.05 for difference between OPG and WT mice as determined by two tailed t test.