Mice Expressing Myc in Neural Precursors Develop Choroid Plexus and Ciliary Body Tumors

Abstract

Choroid plexus tumors and ciliary body medulloepithelioma are predominantly pediatric neoplasms. Progress in understanding the pathogenesis of these tumors has been hindered by their rarity and lack of models that faithfully recapitulate the disease. Here, we find that endogenous Myc proto-oncogene protein is down-regulated in the forebrain neuroepithelium, whose neural plate border domains give rise to the anterior choroid plexus and ciliary body. To uncover the consequences of persistent Myc expression, MYC expression was forced in multipotent neural precursors (nestin-Cre:Myc), which produced fully penetrant models of choroid plexus carcinoma and ciliary body medulloepithelioma. Nestin-mediated MYC expression in the epithelial cells of choroid plexus leads to the regionalized formation of choroid plexus carcinoma in the posterior domain of the lateral ventricle choroid plexus and the fourth ventricle choroid plexus that is accompanied by loss of multiple cilia, up-regulation of protein biosynthetic machinery, and hydrocephalus. Parallel MYC expression in the ciliary body leads also to up-regulation of protein biosynthetic machinery. Additionally, Myc expression in human choroid plexus tumors increases with aggressiveness of disease. Collectively, our findings expose a select vulnerability of the neuroepithelial lineage to postnatal tumorigenesis and provide a new mouse model for investigating the pathogenesis of these rare pediatric neoplasms.

Figure 1

Persistent Myc expression drives tumorigenesis in choroid plexus (ChP). A and B: Immunohistochemical (A) and immunoblot analyses (B) demonstrate higher Myc expression in embryonic day (E) 8.5 forebrain compared to E10.5 forebrain. Dashed lines indicate division between surface ectoderm, mesenchyme (ME), and neuroepithelium (NE) at E8.5, and division between the developing epidermis/meninges and the neuroepithelium at E10.5. C: Lateral ventricle (LV) and fourth ventricle (4V) choroid plexuses from 8- to 9-week–old wild-type (WT) and Myc-overexpressed (OE) mice. White arrows denote tumorigenic region. Note posterior regionalization of tumor in the LV choroid plexus, whereas tumor develops throughout the 4V choroid plexus. D: Hematoxylin and eosin staining shows choroid plexus carcinoma in the LV and 4V choroid plexuses of Myc-OE mice. Arrows indicate mitoses; the boxed areas in left panels are shown at higher magnification in right panels. E: Ki-67 staining shows high proliferation in choroid plexus tumors in both LV and 4V of Myc-OE mice. F: Tumors in both LV and 4V are variably immunoreactive to renal inward rectifier K⁺ channel (Kir7.1). G and H: The percentage of multiciliated epithelial cells is decreased in both the LV and 4V choroid plexuses in the tumorigenic regions of 8-week–old Myc-OE mice. Solid arrows indicate monociliated cells; open arrows indicate multiciliated cells. Data expressed as means ± SEM (H). ^∗P < 0.05 (Welch corrected t-test). Scale bars: 1 mm (C); 500 μm (D, left); 20 μm (D, right; E and F); 10 μm (G). A, anterior; Actb, β-actin; Arl13b, ADP-ribosylation factor-like protein 13b; L, lateral; M, medial; P, posterior.

Figure 2

Nestin-Cre recombination and Myc expression in choroid plexus (ChP). A–H: Nestin-Cre recombination in lateral ventricle (LV; left) and fourth ventricle (4V; right) choroid plexuses shown by red-to-green transition in the Rosa^mTmG mouse strain at postnatal day (P) 0 (A and B), P8 (C and D), and P14 (E–H). Boxed areas in left panels are shown at higher-magnification in right panels. Arrows denote recombined epithelial cells. The boxed areas in A–H correspond to the images at the right at higher magnification. I and J: Myc-positive staining nuclei in tumorigenic regions of posterior LV (I) and 4V (J) choroid plexuses of Myc-overexpressed (OE) mice. Scale bars: 100 μm (A–H, left); 50 μm (A–H, right); 20 μm (I and J). 3V, third ventricle; Ant., anterior; Post., posterior; WT, wild-type.

Figure 3

Myc-driven tumorigenesis up-regulates protein biosynthetic machinery. A and B: Nucleolar volume was higher in Myc-overexpressed (OE) choroid plexuses (ChP) than in those of wild-type (WT) mice at 8 weeks in both the lateral ventricle (LV) and fourth ventricle (4V). Outliers excluded by the ROUT method (Q = 1%). Arrows indicate fibrillarin-positive nucleoli. C: Quantitative RT-PCR measures changes in expression of ribosomal subunits (Rps17, Rps5, Rps12, Rpl11, and Rpl10a) in the choroid plexuses of 7- to 9-week–old Myc-OE mice. Note: LV choroid plexus samples contained entire tissue, including nontumorigenic anterior domain. D:O-propargyl-puromycin (OPP) incorporation into WT and Myc-OE tissue. The boxed areas correspond to the insets shown at higher magnification. E: Overall distribution of cells with high protein-synthesis levels, measured by OPP incorporation, was redistributed to higher OPP levels in Myc-OE choroid plexuses than in those of WT mice at 8 weeks in both the LV and 4V choroid plexuses. Data are expressed as means ± SEM (B, C, and E). n = 4 embryos of each genotype from two litters (C). ^∗∗∗∗P < 0.0001 (U-test); ^†P < 0.05, ^††P < 0.01 (Kruskal-Wallis test). Scale bars: 10 μm (A); 100 μm (D). RPL, ribosomal protein L; RPS, ribosomal protein S.

Figure 4

Human choroid plexus tumors display increased Myc expression correlating with increased proliferation. A: Myc staining in control choroid plexus (top), choroid plexus papilloma (CPP; middle), and choroid plexus carcinoma (CPC; bottom) from patients. Arrowheads indicate Myc-positive cells. B: Quantification of percentage of Myc-positive staining epithelial cells in control choroid plexus, CPP, and CPC samples. C: Representative images showing Myc and Ki-67 staining in control choroid plexus (top), CPC-expressing Myc (middle), and CPC not expressing Myc (bottom) from human patients. Black arrowheads indicate Myc-positive cells; white arrowheads indicate Ki-67–positive cells. D: Ki-67 levels in the Myc-expressing CPC sample are significantly higher than in either the Myc-negative CPC sample or the control samples. Each data point represents one field of view from a single sample. Data are expressed as means ± SEM (B and D). ^∗P < 0.05, ^∗∗∗∗P < 0.0001 (Welch corrected t-test). Scale bars = 20 μm (A and C).

Figure 5

Persistent Myc expression drives tumorigenesis in the eye. A: Gross morphology shows an increase in eye size in the tumorigenic eye of an 8-week–old Myc-overexpressed (OE) mouse as compared to a wild-type (WT) littermate. B and C: Hematoxylin and eosin (H&E) staining shows incipient tumors present in situ arising from the ciliary body of the eye at 4 weeks after persistent Myc expression. The boxed area in B is shown at higher magnification in C. D and E: H&E staining shows medulloepithelioma in 8-week–old Myc-OE mice. The boxed area in D is shown at higher magnification in E. F: Immunohistochemical analysis indicates that the tumor is neither retinoblastoma [absent cone-rod homeobox (CRX), variable synaptophysin], nor melanoma [variable positivity for human melanoma black (HMB)-45, low S100]. G: Ki-67 staining shows high proliferation in the ciliary body medulloepithelioma in Myc-OE mice. The boxed area in G is shown at high magnification in H. H and I: Nucleolar volume was higher in the ciliary bodies of Myc-OE mice than in those of WT mice at 8 weeks, as indicated by fibrillarin staining. Arrows in H indicate fibrillarin-positive nucleoli. Data are expressed as means ± SEM (I). ^∗∗∗∗P < 0.0001 (Welch corrected t-test). Scale bars: 2 mm (A); 1 mm (B and D); 100 μm (C and G); 40 μm (E); 20 μm (F); 10 μm (G, inset, and H). RPE, retinal pigment epithelium.

Figure 6

Cre recombination and Myc overexpression in the eye. A and B: Nestin-Cre recombination in the eye of an 8-week–old (wko) mouse, shown by red-to-green transition in the Rosa^mTmG mouse strain. The boxed area in A is enlarged in B to show the ciliary body of the eye. C: Myc-positive stained nuclei in tumorigenic regions of ciliary body of the eye in Myc-overexpressed (OE) mice. Scale bars: 100 μm (A); 50 μm (B); 20 μm (C). WT, wild-type.

Supplemental Figure S1

Positron emission tomography does not reveal ¹⁸F fluorodeoxyglucose (FDG)–positive Myc-driven tumors in the body. Representative examples of PET imaging with FDG at 8 weeks old (wko) does not reveal FDG-positive tumors in the bodies of the mice. Arrows denote brain region. Asterisks denote bladder tissue where FDG is detected in normal-excretion pathway and is not associated with tumor formation. Similar results are observed up to 10 weeks of age. OE, overexpressed.