Published OnlineFirst September 22, 2017; DOI: 10.1158/0008-5472.CAN-17-1018 Cancer Molecular and Cellular Pathobiology Research
Cic Loss Promotes Gliomagenesis via Aberrant Neural Stem Cell Proliferation and Differentiation Rui Yang, Lee H. Chen, Landon J. Hansen, Austin B. Carpenter, Casey J. Moure, Heng Liu, Christopher J. Pirozzi, Bill H. Diplas, Matthew S. Waitkus, Paula K. Greer, Huishan Zhu, Roger E. McLendon, Darell D. Bigner, Yiping He, and Hai Yan
Abstract
Inactivating mutations in the transcriptional repression oligodendrocyte differentiation. Cic is known to participate in factor Capicua (CIC) occur in approximately 50% of human gene suppression that can be relieved by EGFR signal, but we oligodendrogliomas, but mechanistic links to pathogenesis found that cic also activated expression of a broad range of are unclear. To address this question, we generated Cic-defi- EGFR-independent genes. In an orthotopic mouse model of cient mice and human oligodendroglioma cell models. glioma, we found that Cic loss potentiated the formation and Genetic deficiency in mice resulted in a partially penetrant reduced the latency in tumor development. Collectively, our embryonic or perinatal lethal phenotype, with the production results define an important role for Cic in regulating neural of an aberrant proliferative neural population in surviving cell proliferation and lineage specification, and suggest mech- animals. In vitro cultured neural stem cells derived from Cic anistic explanations for how CIC mutations may impact the conditional knockout mice bypassed an EGF requirement for pathogenesis and therapeutic targeting of oligodendroglioma. proliferation and displayed a defect in their potential for Cancer Res; 77(22); 1–12. 2017 AACR.
Introduction are exclusively restricted to oligodendrogliomas, suggesting a possible link of CIC and FUBP1 to oligodendrocyte lineage in Oligodendrogliomas are a histologic type of diffuse glioma neurogenesis and tumorigenesis. (WHO grade II–III) that account for between 5% and 20% of CIC is a member of the Sox-related high-mobility group gliomas (1) and frequently display characteristic loss of whole (HMG) subfamily and is highly conserved among species (12). chromosome arms 1p and 19q (1p/19q-codeletion; refs. 2–4). Previous evidence suggests that cic acts primarily as a transcrip- Relative to other diffuse glioma subtypes, oligodendrogliomas are tional repressor consisting of two highly conserved domains: the associated with better prognosis due in part to the tumor's HMG box, which mediates DNA binding and nuclear localiza- increased chemosensitivity (5). However, tumor recurrence tion, and a C-terminal motif C1, which co-operates with the remains the most common cause of death for oligodendroglioma HMG-box for DNA binding (12–16). In Drosophila, cic transduces patients, as these tumors often recur or progress to higher grades. the signaling of receptor tyrosine kinases (RTK), Torso, and the Previous studies from ours and other groups have revealed recur- EGFR, and by doing so regulates cell proliferation and cell lineage rent inactivating mutations of Capicua (CIC, on chromosome arm specification during development (13, 15, 17–19). 19q) and far upstream element binding protein (FUBP1,on Besides oligodendrogliomas, CIC is also linked to other cancers chromosome arm 1p), identifying these genes as putative tumor and noncancer diseases in which its function as a transcriptional suppressors for oligodendroglioma (6–9). Somatic mutations of regulator is believed to be involved (14, 20). However, the these two genes, together with IDH1R1/2 mutations, 1p/19q loss, mechanism underlying the role of CIC inactivation in oligoden- and TERT promoter mutations, define oligodendroglioma as a droglioma genesis remains unclear, in part due to the lack of specific brain tumor subtype (6, 7, 10, 11). Interestingly, appropriate animal and cell models of oligodendroglioma. In this whereas TERT promoter and IDH mutation occur across multiple study, we generated Cic knockout mouse models, mouse neural diffuse glioma subtypes, somatic mutations of CIC and FUBP1 stem cell (NSC) lines, and isogenic CIC knockout human oligo- dendroglioma cell lines to investigate the effect of Cic inactivation Department of Pathology and the Preston Robert Tisch Brain Tumor Center at (loss) on neural development and on oligodendroglioma path- Duke, Duke University Medical Center, Durham, North Carolina. ogenesis. We reveal aberrant neural development in vivo and defective NSC differentiation/proliferation in vitro upon Cic Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). knockout, illuminate genes/pathways regulated by Cic via both EGFR-dependent and -independent mechanisms, and present Corresponding Authors: Hai Yan, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center at Duke, 203 Research Drive, MSRB-1, Room direct evidence to support the tumorigenic role of Cic loss in 199B, Durham, NC 27710. Phone: 919-668-7850; Fax: 919-684-8756; E-mail: oligodendroglioma genesis in vivo. [email protected]; and Yiping He, Department of Pathology, Duke University Medical Center, DUMC-3156, 199A-MSRB, Research Drive, Durham, NC 27710. Materials and Methods Phone: 919-684-4760; Fax: 919-684-8756; E-mail: [email protected] fl fl Generation of CICtm2a(KOMP)Wtsi mice and Cic ox/ ox mice doi: 10.1158/0008-5472.CAN-17-1018 KOMP plasmid clone PRPGS00139_B_H08 was transfected 2017 American Association for Cancer Research. into 129/B6 hybrid embryonic stem cells (ESC) by
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electroporation (21). Targeted ESC clones were screened by PCR antibodies, cells were washed three times in PBS and stained and chimeric mice were generated by tetraploid blastocyst com- with appropriate Alexa Fluor secondary antibodies (1:500) for plementation performed by the Duke Transgenic Mouse Facility. 30 minutes at room temperature. Cells were washed with PBS and F0 chimeric mice were further bred to C57BL/6 wild-type mice to counterstained with DAPI before being mounted with mounting þ generate F1 heterozygous Cictm2a(KOMP)Wtsi mice (Cicnull/ ). F1 medium (Vectashield H-1400). Slides were imaged with a Nikon mice were inbred to generate homozygous Cicnull/null mice. In this ECLIPSE TE2000-E fluorescent microscope. case, viable homozygous Cictm2a(KOMP)Wtsi offspring were under- represented due to the partial embryonic lethality. F1 mice were In vitro proliferation assay and drug study then bred to Flp-expressing mice (FLPo-10 from The Jackson Cells were plated on laminin-coated 96-well plates at a density Laboratory) to remove the Frt-flanked gene trap cassette in the of 1,000–3,000 cells/well. At designated time points, CCK8 fl þ Cictm2a(KOMP)Wtsi allele, resulting in a conditional allele (Cic ox/ ; reagent (Dojindo, CK04-20) was added into each well according fl þ ref. 21). Further inbreeding of these Cic ox/ mice allowed gen- to manufacturer's instructions. After incubation for 3–5 hours, fl fl eration of homozygous Cic ox/ ox mice, which allow further OD450 absorbance of each well was screened by a microplate conditional knockout in the presence of Cre expression. reader (Tecan, infinite M200PRO). For cell response to U0126, cells were plated out for 24 hours before U0126 was added into Genotyping the wells. Tissue was lysed in lysis buffer (100 mmol/L Tris-HCl pH 8; 5 mmol/L EDTA pH 8; 0.2% SDS; 200 mmol/L NaCl; Proteinase Gene expression microarray analysis þ þ K, and water) and DNA was ethanol precipitated. Pellet was Four Cic / and three Cic / neonatal NSC lines (established m resuspended in 100 L sterile H2O. One to two microliters of from different animals independently) were cultured in 6-well diluted DNA were used for PCR (15 mL reaction volume). PCR was plates in the standard proliferation medium (StemCell Technol- performed as followed: 95 C for 5 minutes, 30 cycles of (95 C for ogies; catalog no. 05702) with or without EGF (20 ng/mL) for 15 seconds, 60 C for 15 seconds, 72 C for 15 seconds), 72 C for 24 hours. RNA was then extracted by RNA Miniprep Kit (Zymo; 5 minutes. Genotyping primers can be found in Supplementary catalog no. 11-328) following the manufacturer's instruction and Table S1. FFPE genomic DNA was extracted by the GeneRead DNA submitted to Duke Sequencing and Genomic Technologies FFPE Kit (Quagen; catalog no. #180134) according to the man- Shared Resource for microarray hybridization (Affymetrix Gene- ufacturer's instructions, and further genotyped to exclude chime- Chip MO 2.0 ST; catalog no. 902118). Gene expression micro- ric expression of cic in brain tissue. array data have been submitted to the Gene Expression Omnibus database under accession GSE95012. The Robust Multichip Mouse neonatal NSCs and virus transduction Average (RMA) method was applied for data processing and fl fl þ þ Postnatal day 4 (P4) Cic ox/ ox or Cic / neonatal mice were normalization by R. For the most differentially expressed genes anesthetized by hypothermia and then decapitated with sharp in response to EGF depletion, fold changes of gene expression þ þ scissors. The subventricular zone (SVZ) was microdissected from (EGF vs. EGFþ) of both Cic / and Cic / cells were calculated brains and dissociated into single cells by trituration. These cells and averaged (designated as fold change 1 and fold change 2), and were then cultured as neurospheres in EGF/FGF containing difference of fold changes 1 and 2 (Dfold change) were calculated. þ þ serum-free neural stem cell medium (StemCell Technologies; Most differentially expressed genes in Cic / versus Cic / NSCs catalog no. 05702) following the manufacturer's instructions. To in response to EGF depletion were selected if Dfold change (log2 fl fl obtain Cic / NSC lines, Cic ox/ ox NSC lines were transduced ratio) was equal or greater than 1. The enrichment score is log with Cre-expressing adenovirus (Vectorbiolabs; catalog no. 1769) (P value or EASE score). EASE score (or P value) is a modified at a multiplicity of infection (MOI) of 10–100. Removal of the Fisher exact test calculated by the same method as pathway þ þ stop cassette was verified by PCR. Cic / NSC lines were similarly analysis by DAVID (22). To Stratify genes regulated differently transduced with the Cre-expressing adenovirus and served as the by EGFR and/or Cic, genes with expression absolute fold change þ þ þ þ control (Cic / NSC lines). These cells were used for all in vitro by EGF (EGF vs. EGFþ,inCic / cells) equal or greater than 2 þ þ assays (proliferation assay, drug resistance assay, and microarray). and absolute fold change by Cic (Cic / vs. Cic / ) in EGF- depleted condition equal or greater than 2 were defined as In vitro differentiation of NSCs and immunofluorescent EGF-regulated Cic downstream targets (group I, 44 genes); genes staining with expression absolute fold change by EGF (EGF vs. EGFþ,in þ þ þ þ Cic / or Cic / NSCs were cultured for 7 days in 8-chamber Cic / cells) equal or greater than 2 and absolute fold change by þ þ vessel glass slides (BD Falcon; ref. no. 354108) in either complete Cic (Cic / vs. Cic / ) in both EGFþ and EGF-depleted condi- proliferation medium (StemCell Technologies; catalog no. tions smaller than 2 were defined as non-Cic–related EGF down- 05702) supplemented with EGF (20 ng/mL) and FGF (10 ng/mL) stream targets (group II, 183 genes); genes with expression abso- þ þ or differentiation medium (StemCell Technologies; catalog no. lute fold change by Cic (Cic / vs. Cic / ) in EGF-depleted 05704). Cells were fixed by formalin, blocked with blocking condition equal or greater than 2 and absolute fold change by þ þ buffer (1% BSA, 10% goat serum in PBS), and stained with the EGF (EGF vs. EGFþ, in both Cic / and Cic / cells) smaller following primary antibodies: Nestin (StemCell Technologies; than 2 were defined as non-EGF–regulated Cic downstream catalog no. 60051, 1:50), GFAP (StemCell Technologies; catalog targets (group III, 192 genes). Cell type–specific genes are defined no. 60048, 1:200 or Cell Signaling Technology; catalog no. 12389, according to http://www.stanford.edu/group/barres_lab/brain_r 1:100), Olig2 (Millipore; catalog no. AB9610, 1:100), Ki67 (BD; naseq.html (23). Fold enrichment of astrocytes, neurons, newly catalog no. 550609, 1:100), b3 tubulin (StemCell Technologies; formed oligodendrocytes, myelinating oligodendrocytes, OPC, catalog no. 01409, 1:1,000), or CNPase (Cell Signaling Technol- microglial, and endothelial cells are calculated as FPKM of one cell ogy; catalog no. 5664, 1:100). After blocking with primary type divided by average of FPKM of all other cell types. Top 500
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candidate genes of each cell type were documented. Genes specific trols (IgG control and CIC knockout cell line control, respectively) to "newly formed oligodendrocytes" and "myelinating oligoden- by MACS2 2.1.1. Narrow regions of enrichment were identified drocytes" are merged as "oligodendrocytes" for simplicity. using MACS2 2.1.1 with the fragment size of 300 bp based on wet experiment results and the q-value cutoff as 0.05 (http://github. Generation of isogenic CIC knockout HOG cell lines by com/taoliu/MACS/; ref. 31). ChIP-seq signal track was visualized CRISPR-Cas9 by the Integrative Genomics Viewer (IGV; refs. 32, 33). Peak A human oligodendroglioma cell line (HOG) was cultured in location data were annotated by PAVIS (34). Iscove's Modified Dulbecco's Medium (Gibco, Invitrogen) sup- plemented with 10% FBS as previously described (24). This cell Gliomagenesis modeling via orthotopic implantation of NSCs þ þ line is wild type in both IDH1 and IDH2 genes and does not have Cic / and Cic / NSC lines (one line for each genotype) were 1p/19q codeletion. The CRISPR/Cas9 system was used for gene transduced with retrovirus expressing human platelet-derived editing. pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng growth factor subunit B (PDGFB). The retrovirus construct, Zhang (Addgene plasmid No. 48138; ref. 25). Double-stranded MigR1-PDGFB, coexpresses GFP and PDGFB from the same oligonucleotides were cloned into restriction enzyme BbsI-line- transcript via an internal ribosomal entry site (IRES; Addgene no. arized PX458 to construct plasmids for CRISPR/Cas9 targeting of 27490; ref. 35). MigR1-PDGFB retrovirus particles were produced human CIC. Three individual sgRNA sequences targeting human in 293FT cells, and used to transduce NSCs at an MOI of 10–100. CIC were designed with optimal targeting efficiency and minimal Transduced NSCs were further sorted by FACS (BD FACSVantage þ off-targets (26, 27), and one nontargeting sequence (NTC; ref. 28) SE cell sorter, Duke Cancer Institute) for GFP cells. The resulting þ þ þ was used as a negative control. The sequences of the sgRNAs are: GFP /PDGFB–expressing Cic / or Cic / NSCs were injected sgRNA-e1: gctactgaccgaacatgccg, sgRNA-e4: ctctaccgcccggaaaacgt, intracranially into the right striatum of 4- to 6-week-old NSG mice sgRNA-e13: agagctcgtgggcccgtacg, sgRNA-NTC: gtatcctgacctacgcg- at a dose of 2.5 105 cells per mouse. Mice were sacrificed when ctg. For targeting CIC, three different combinations of sgRNAs they showed signs or symptoms of brain abnormalities such as (e1þe4, e1þe13, e4þe13) were used for gene knockout via allele macrocephaly or lethargy. Brains were harvested, formalin-fixed, deletions or indels. For transient plasmid transfection, plasmids and embedded in paraffin for subsequent IHC and immunoflu- (two plasmids at 1:1 ratio for achieving the desired gene deletion/ orescence staining assays. mutations) and Transfex (ATCC; catalog no. ACS-4005) were mixed and used for cell transfection according to manufacturer's instruc- The Cancer Genome Atlas data analysis þ tions. One to three days after the transfection, GFP cells were All The Cancer Genome Atlas (TCGA) gene expression data sorted via FACS (BD FACSVantage SE cell sorter, Duke Cancer were downloaded from the online portal, https://gdc-portal.nci. þ Institute) to obtain a GFP population to enrich transfected cells. nih.gov/. CIC copy number and mutation status, as well as These cells were further cultured for 1 to 3 days allowing them to IDH1/2 mutation status and histologic subtype were retrieved recover from the FACS and then serially diluted in 96-well plates for from cBioportal (36, 37), and matched to cases with gene expres- generating single-cell clones. Clones were screened for inactivating sion data available. The 2016 TCGA Merged Glioma cohort was alterations by Sanger sequencing. Four subclones bearing different used for all analyses (38). All patient cases with complete infor- homozygous nonsense mutations in CIC were identified and mation available (i.e., confirmed genotype of IDH, CIC) were numbered as HOG_1, HOG_2, HOG_3, and HOG_4, respectively. used for each analysis unless otherwise stated, which consisted Ablation of CIC protein level (both long isoform of CIC, CIC-L, and of 100 oligodendroglioma patients. A total of 748 genes were short isoform of CIC, CIC-S) was validated by CIC Western blot generated for heatmap by using a t test to compare expression þ analysis (Novus Biologicals; catalog no. NB110-59906, 1:1,000). levels of each gene between CIC / and CIC / cases and filtering Short tandem repeat (STR) profiling was performed by the Cell for all genes with a t test P <2.5 10 3. Culture Facility at Duke to validate that the four isogenic CIC knockout lines have consistent STR profiling with the parental line. Statistical analysis þ þ þ Quantification of Olig2 cells in the adult survival Cic / and Chromatin immunoprecipitation sequencing and data analysis Cic / mice were manually performed using two slides from each Chromatin immunoprecipitation (ChIP) was performed as animal (n ¼ 2 for each genotype) and a two-tailed unpaired Student described previously (29). Briefly, cells were cross-linked and cell t test was used to assess significant differences between conditions. lysate was then prepared and subjected to sonication to shear the For all in vitro proliferation assays by CCK8, at least two independent chromatin. Protein G magnetic beads (New England BioLabs; cell lines of each genotype were included and experiments were catalog no. S1430S) conjugated with anti-CIC antibody (Novus conducted at least twice. Two-tailed unpaired Student t test analysis Biologicals; catalog no. NB110-59906) were used for immuno- was performed to compare the Cic / lines to the control lines precipitation. ChIP-derived DNA was recovered for library prep- (three technical replicates were performed for each line). qPCR for aration. High-throughput sequencing (Illumina HiSeq 4000 sys- gene expression analysis was performed with two technical repli- tem) was performed by Duke Sequencing and Genomic Technol- cates and the DDCt method to determine normalized gene expres- ogies Shared Resource, and data analysis was performed by Duke sion. For Kaplan–Meier curve, log-rank test P value was calculated. Genomic Analysis and Bioinformatics Shared Resource. ChIP-Seq For all microarray data analysis, expression absolute fold change data has been submitted to Sequence Read Archive database cutoff was set to equal or greater than 2. For all experiments, error under the accession number SRP119146. Reads were mapped to bars represent SE; , P < 0.05; , P < 0.01; , P < 0.001. GRCh37 version of the human genome using the Bowtie align- ment algorithm (30). For the aligned data, signal tracks were Ethics statement generated according to the calculation of the log 10 likelihood All animal studies were approved by the Duke Institutional ratios between CIC wild-type line ChIP-seq signal and two con- Animal Care and Use Committee, an institution accredited by the
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Association for Assessment and Accreditation of Laboratory pups, only 6 (7.5%) of them, including one runt that died shortly Animal Care (AAALAC), International. Protocol number: A072- after birth, were homozygous knockout, likely due to a partial 13-03 and A037-16-02. embryonic or perinatal lethality in Cicnull/null animals. We first determined the effect of Cic knockout on neural development by examining the brain from viable, postnatal Results Cicnull/null animals. As expected, at postnatal day 4 (P4), both þ þ Knockout of Cic results in aberrant hyperproliferative cells in wild-type control (Cic / ) and Cicnull/null mice displayed active the adult SVZ postnatal neurogenesis, as validated by the expression of Ki67 Previous studies have revealed the essential role of CIC in around the lateral ventricles (Supplementary Fig. S2). By P28, development and the neurodegeneration disorder SCA1 in hu- although Ki67 expression was no longer detected in the SVZ in mans (20). To determine the effects of a Cic inactivation in neural wild-type animals, as expected (39), active cell proliferation (Ki- development, we generated a Cic knockout mouse model using a 67-positive) was readily detectable in Cicnull/null mice (Fig. 1B). targeting plasmid, Cictm2a(KOMP)Wtsi (Fig. 1A; Supplementary Furthermore, the population of GFAP-expressing cells was greatly Fig. S1A; ref. 21). Heterozygous founder mice were crossed elevated in the SVZ of Cicnull/null mice compared with those in the to obtain homozygous Cic knockout progeny in which both wild-type mice (Fig. 1C). As GFAP-expressing type B radial cells alleles are Cictm2a(KOMP)Wtsi (abbreviated as Cicnull/null hereafter) represent the dominant NSC population in the SVZ (40), these (Supplementary Fig. S1B). In addition, conditional knockout results suggest Cic loss leads to an aberrant hyperproliferative fl fl Cic ox/ ox mice can be generated by breeding F1 mice to Flp- population in the adult SVZ. Consistent with these hyperproli- expressing mice (Fig. 1A; Supplementary Fig. S1C). Among 80 live ferative phenotypes, DAPI staining of the brain tissue sections at
Figure 1. Cic knockout results in aberrant hyperproliferative cells in adult SVZ. A, Cic knockout strategy. A gene trap cassette flanked by FRT sites was inserted between Cic exon 8 and 9, and exon 9, 10, and 11 were flanked by loxP sites. When treated with Flp recombinase, the gene trap cassette can be removed, resulting in a conditional allele (Cic function is restored). This conditional allele can be further converted into a knockout allele by deleting exon 9, 10, and 11 via Cre recombinase. Gray arrows, genotyping primers. B–E, Representative immunofluorescent stained FFPE slides from similar planes of postnatal day 28 (P28) Cicþ/þ (n ¼ 2) and Cicnull/null (n ¼ 2) mouse brains for the following markers: Ki67 ( 10; B), GFAP ( 10; C), Dapi ( 20; D), and Olig2 ( 10; E). F, Quantifications of Olig2þ cells as shown in E. En2 SA, mouse En2 splicing acceptor; IRES, internal ribosome entry site; LacZ, LacZ reporter; pA, SV40 polyadenylation sequences; hbactP, human b-actin promoter; CC, corpus callosum; LV, lateral ventricle; LSD, lateral septal nucleus.
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Figure 2. Cic knockout confers EGFR signaling–independent propagation of NSCs. A, Propagation of two Cicþ/þ NSC lines under mild hypoxic culture condition (3% oxygen) and with or without EGF. B, Propagation of two Cic / NSC lines under the same experimental condition as in A. C, Propagation of Cicþ/þ and Cic / NSC lines in the presence of U0126 for 5 days. Cell numbers of each cell line were normalized to their DMSO-treated controls. similar planes revealed thicker lateral ventricular walls in proliferation medium greatly attenuated the expansion of the þ þ Cicnull/null mice compared with those in Cic / mice (Fig. 1D). NSCs, regardless of their Cic status (Supplementary Fig. S3A and Finally, although no differential expression of Nestin or Sox2 S3B). Although hypoxia in most cases is considered to play an þ þ were observed in the SVZ of Cic / and Cicnull/null mice, there was important role in pathologic conditions such as tumorigenesis, it a significantly increase in the number of cells expressing Olig2 is also well established that a physiologic gradient of oxygen exists þ (Olig2 ) in the dorsal part of lateral septal nucleus (LSD) of in the central nervous system (CNS) and that mild hypoxia is a Cicnull/null mice (Fig. 1E and F), suggesting an aberrant expansion/ physiologic condition in normal brains (41). We therefore tested migration of the oligodendrocyte progenitor cells (OPC) and the proliferation of NSCs under a mild hypoxic culture environ- providing a potential link between Cic inactivation and oligo- ment (3% oxygen). We found that under the standard, EGF- þ þ dendrocyte differentiation. Collectively these results suggest supplemented proliferation media, both Cic / and Cic / NSCs that Cic inactivation results in aberrant development/expansion again propagated robustly (Fig. 2A and B). However, when EGF of cell populations in the adult SVZ, reminiscent of a preneoplas- was withdrawn from the media, a striking difference in response þ þ tic phenotype. was observed: although the growth of Cic / NSCs was complete- ly attenuated, the growth of Cic / NSCs was barely affected by Cic / NSCs bypass the requirement of EGF for proliferation the EGF withdrawal (Fig. 2A and B; Supplementary Fig. S3C and under hypoxia condition S3D). In agreement with this resistance to EGF withdrawal, Cic / The observed preneoplastic phenotype raises the possibility NSCs were more resistant to U0126, an inhibitor of the down- that Cic knockout leads to aberrant proliferation of NSCs and/or stream kinase of EGFR signaling, MEK (Fig. 2C). These results are other neural lineage progenitor cells. To overcome the high consistent with previous findings showing that cic acts as a mortality seen in generating Cicnull/null animals, we crossed the downstream mediator of the EGFR signaling pathway (13, 17, þ F1 mice (Cicnull/ ) with FLPase-transgenic mice (FLPo-10 from 19). In addition, they provide one potential mechanism under- the Jackson Laboratory) to achieve Flp-mediated deletion of the lying the aberrant proliferation of NSCs in vitro and the preneo- gene trap cassette. These resultant mice contained functional Cic plasia we observed in vivo. The EGF-independent growth of Cic / fl alleles (Cic ox), which allowed for subsequent, Cre recombinase- NSCs under the mild hypoxic condition is particularly interesting induced Cic knockout (Fig. 1A). NSC cell lines were established as hypoxia/oxygen concentration is a prominent factor in both þ þ fl fl from the SVZ of P4 Cic / or Cic ox/ oxmice followed by adeno- physiologic and tumorigenic conditions in the CNS (41, 42). virus-mediated transient Cre expression, resulting in the genera- / tion of Cic / NSC lines (Supplementary Fig. S1C). Cic NSCs have a compromised oligodendrocyte We first analyzed the proliferation of NSCs and found that differentiation potential and are arrested in an OPC-like state under the standard NSC proliferation medium condition (EGF- The aberrant NSC proliferation observed in the above models, þ þ supplemented, normoxic culture), both Cic / and Cic / NSCs together with the knowledge that CIC's inactivating mutations propagated robustly. As expected, the growth of the NSCs was predominantly occur in human oligodendrogliomas (6–9), dependent on the EGFR signaling, as withdrawal of EGF from the prompted us to investigate the effect of Cic knockout on neural
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Figure 3. Cic / NSCs fail to differentiate into mature oligodendrocyte and are arrested in an OPC-like state. Cicþ/þ and Cic / NSCs were maintained in the standard proliferation media or in differentiation medium for seven days. Immunofluorescent staining was performed. A, Staining ( 10) for Olig2 and Dapi. B, Quantification of Olig2þ cells upon differentiation in A. Three randomly chosen fields were quantified for each cell line (two-tailed t test analysis). C, Postdifferentiation staining ( 10) for GFAP and CNPase and counterstained with Dapi. D, Most differentially expressed genes in Cic / versus Cicþ/þ NSCs in response to EGF depletion. Each column represents fold change (EGF vs. EGFþ) of gene expression for a sample. Positive value (red) represents higher expression in EGF-depleted condition compared with EGF-supplemented condition. Genes defined as different neural lineage markers are color-coded on the left. Enrichment scores [ log(P-value or EASE score)] were calculated for each lineage marker (see Materials and Methods for details). E, A proposed schematic for Cic loss-induced compromised oligodendrocyte differentiation.