Cross-Species Genomics Identifies TAF12, NFYC, and RAD54L as Choroid Plexus Carcinoma Oncogenes

Abstract

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Figure 1. A mouse model of CPC

Expression of the recombined ROSA^YFP fluorescent lineage tracing allele in whole (A) and microscopic (B) preparations of postnatal day (P) 0 hindbrain choroid plexus following in utero electroporation with Cre-recombinase at embryonic day (E) 12.5 (scale bar=50μm). Recombined (C), but histologically normal (D) and Ttr⁺ (E) choroid plexus persisting in adult CPE (scale bar=15μm). In utero electroporation of the hindbrain choroid plexus of Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP E12.5 embryos generated large YFP⁺ tumors (F,G) that recapitulate the histology (H); reduced Ttr (I) and increased Cytokeratin 8 (J) expression; high proliferation rate (K); and ultrastructural features (L,M) of human CPC (scale bar F to K=20μm; L to M=2μm). N, polymerase chain reactions of recombined (RC) alleles in mouse CPC (T) and intact (WT) alleles in normal tissue (N). O, Kaplan-Meier survival curves of E12.5 mice harbouring the indicate alleles that were Cre-electroporated in utero. P, unsupervised hierarchical clustering of gene expression profiles of mouse: CPC; E12.5 and adult CPE (eCP and aCP); WNT and Sonic Hedgehog (SHH) medulloblastomas; P7 dorsal brainstem (P7 DBS); P7 cerebellum (P7 CB); E12.5 cerebellum (eCB); and E12.5 lower rhombic lip (eLRL). Q, GSEA of ‘Lein choroid plexus markers’ in CPC reporting normalized enrichment score (NES) and the false discovery rate (FDR) Q value. See also Table S1.

Figure 2. Cross-species analysis of syntenic chromosomal gains in human and mouse CPC

Heat maps of genomewide DNA copy number alterations in human CPPs and CPCs (A) and mouse CPCs (B). Heatmaps of the copy number of chromosome 1 (C), 7 (D) and 12 (E) in human CPPs and CPCs (left in each figure) and the corresponding syntenic regions in mouse CPCs (right in each figure). See also Tables S2–7.

Figure 3. A common set of 21 syntenic genes gained and overexpressed on 1p31.3-ter in human and 4qC6-qE2 in mouse, CPC

Top: heat maps of human chromosome 1 copy number in 34 human CPCs and CPPs (left), and mouse chromosome 4qC6-qE2 in 27 mouse CPCs and normal choroid (right). Middle: TP53 status and tissue type of each sample. Bottom: heat maps reporting the expression of 21 copy-number driven orthologs located on human chromosome 1p31.3-ter in the 34 human tumors (left) and 27 mouse CPCs and normal choroid (right). Dotted lines demarcate the results of independent microarray probes for each human and mouse ortholog. Gene symbols shown right. See also Figure S1.

Figure 4. Serial analysis of choroid plexus transformation in mice

A, In utero electroporation of the hindbrain choroid plexus of Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP E12.5 embryos resulted in progressive expansion of YFP⁺ choroid plexus (top row, scale bar=50μm) that displayed: dysplasia (second row, hematoxylin and eosin [H&E]; dotted line and arrows indicate disruption of normal single layer epithelium; scale bar=10μm); loss of Ttr expression (third row, in situ hybridization; scale bar=20μm) and increasing proliferation (fourth row, nuclear BrdU incorporation marked with arrows [note dotted line encompasses same region in P0 H&E stain] scale bar=10μm); accumulation of DNA DSB (γH2ax stain marked with arrows; scale bar=10μm); and gain of 4qC6-qE2 (note two separate FISH probes used targeting Stmn1 and Cdca8 relative to control Baat at 4qB1). Graphs to the right report the quantification of BrdU incorporation (B), γH2ax stain (C) and gain of 4qC6-qE2 (D) in recombined (YFP⁺) cells. Mann Whitney, *= P<0.05, ***= P<0.0005.

Figure 5. Functional in utero assessment of 1p31.3-ter/4qC6-qE2 candidate CPC oncogenes

A. E12.5 choroid plexus was co-electroporated with plasmids encoding candidate and GFP to identify electroporated cells. Gels below show seven examples of 21 reverse-transcriptase (RT) PCR results of cells transfected with control plasmid or oncogene candidates with or without RT. B. Sections from each animal were analysed both for morphological change (top, H&E) and proliferation of electroporated cells (bottom, BrdU⁺/GFP⁺; scale bar=10μm). Only Taf12, Nfyc and Rad54l demonstrated dysplasia and aberrant proliferation relative to the other 18 candidates, Pgd shown as an example. C, Graph reporting the percentage of electroporated (GFP⁺) and proliferating choroid plexus epithelium cells. *P<0.05, **=P<0.005, Mann-Whitney.

Figure 6. Taf12, Nfyc and Rad54l are required to initiate and maintain CPC in mice

A. Reverse-transcriptase PCR of CPC cells transduced with control or gene targeted shRNA lentivirus. B. In vitro survival of CPC cells transduced with the indicated shRNA lentivirus. ***=P<0.0005, relative to control^shRNA cells. C. Caspase 3/7 assays of control and shRNA-transduced CPC cells. *=P<0.05, relative to control^shRNA cells. D. Kaplan-Meyer survival curves of mice implanted with CPC cells transduced with the indicated shRNA lentiviruses. *=P<0.05, **=P<0.005, ***=P<0.0005, Log-Rank relative to control^shRNA mice. E. Kaplan-Meyer survival curves of Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP mice electroporated at E12.5 with Cre-recombinase and simultaneously injected in the hindbrain with the indicated shRNA (P<0.05, Log-Rank comparison of the three survival curves). F. Morphology (H&E), lentiviral transduction (GFP), and Cre-recombination (Rosa^YFP) of hyperplastic choroid plexus mass in adult Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP mouse electroporated at E12.5 with Cre-recombinase and simultaneously injected in the hindbrain with Taf1^shRNA (scale bar=20μm).

Figure 7. Taf12, Nfyc and Rad54l expression promote CPC in mice

A, Kaplan-Meyer survival curves of Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP mice injected in the hindbrain at E12.5 with Taf12, Nfyc and Rad54l expressing lentiviruses following sham (no Cre) or Cre-electroporation (Cre). Survival of Cre-electroporated mice not receiving lentivirus is also shown (Cre only). B, Left: macroscopic, direct YFP fluorescence of the hindbrains of adult Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP mice receiving hindbrain injections of the indicated lentivirus at E12.5. Right panels show images of H&E stained or immunofluorescence stained sections of the corresponding hindbrain, left (scale bars=50μm). Kaplan-Meyer survival curves of Tp53^flx/flx; Rb^flx/flx; Pten^flx/flx; ROSA^YFP mice injected in the hindbrain at E12.5 (C) or P1 (D) with the indicated lentiviruses. Statistics report the Log-Rank comparison of the survival curves. D, in situ hybridization (top and middle) and immunofluorescence (bottom) of Taf12, Nfyc and Rad54l expression in CPCs induced by lentiviral injection. All scale bars=50μm.

Figure 8. DNA repair is upregulated in CPC

A. GSEA of ‘Kauffmann DNA Repair Gene’ set in CPC versus E12.5 CPE. B. Heat map reporting the GSEA of the ‘Kegg Homologous Repair Gene’ set in CPC versus E12.5 CPE. C. Growth inhibition assays of mouse CPC and ependymoma (Johnson et al., 2010) cells following 72 hour exposure to the ATR inhibitors AZ-20 and VE-821. D. Quantification of DNA DSBs in the CPC and ependymoma cells shown in C, both before (time 0) and at the indicated times following exposure to 0.4μM AZ-20. See also Table S8.