Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner

Abstract

Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g., SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome.

Figure 1.

GNS cells can be converted to an iPSC-like state. (A) GNS cell lines G7 and G26 were established from tumor samples obtained from two different patients. (Left panels) Original tumors show typical GBM histopathology (H&E) and GFAP immunoreactivity. G7 and G26 grow as adherent cell lines and are positive for the immature neural progenitor markers SOX2 and NESTIN. (Right panels) Upon xenotransplantation, they form tumors similar to the original patient tumor. (B) Strategy used for epigenetic reprogramming of GNS cells. Cells (2 × 10⁶ to 6 × 10⁶) were transfected with piggyBac vectors (KLF4 and OCT4 driven by a CAG promoter). Hygromycin selection was applied for at least 3 wk. Medium was changed to hESC condition after 1 wk. (C) Colonies resembling typical hESC colonies emerged after 4–7 wk for iG7, iG26, and control NS cells (iCB660). iG2 and iG144 were less well defined. Shown are typical examples after clonal colony picking and initial passaging (P3). (D) qRT–PCR for the critical pluripotency marker gene NANOG and the neural marker gene GFAP. Following reprogramming, these genes are activated and suppressed, respectively, and reach levels similar to control iPSCs or hESCs. SOX2 is expressed by ESCs, iPSCs, and NS cells. Levels were normalized to GAPDH. RNA was derived from iPSC and GiPSC culture passages 4–8. (E) Correlation analysis of hESCs, iPSCs, NS cells, GNS cells, and GiPSCs, based on expression levels of 90 markers present on the TLDA (Applied Biosystems) pluripotency panel. iG7 and iG26 are more similar to iPSCs and hESCs than to corresponding parental GNS cells or normal NS cells (CB660). iG2 and iG144 likely represent incompletely reprogrammed lines and failed to correlate with iPSCs/hESCs. RNA was derived from iPSC and GiPSC culture passages 4–8. See also Supplemental Figure 1.

Figure 2.

Gene expression profiling and marker analysis confirms that iG7 and iG26 are reprogrammed to a hESC/iPSC state. (A) Immunocytochemistry for pluripotency marker NANOG and cell surface markers (SSEA4, TRA1-60, TRA2-49, TRA1-81, and TRA2-54). All tested iG7 and iG26 clonal cell lines (iG7-1, iG7-2, iG7-3; iG26-1, iG26-2, and iG26-3; P4–P10) were immunopositive for these pluripotency markers (iG7-1 and iG26-1 are shown), whereas parental GNS lines G7 and G26 were negative. SSEA4 immunostaining is shown in live cells. (B,C) Genomic analysis using Affymetrix SNP 6.0 microarrays for GNS cells (red) and their reprogrammed derivatives (GiPSCs, P8–P12; blue and green) identifies many hallmark genetic changes common to GBM, such as amplification of chromosomes 7, 19, and 20 (arrows) and losses of chromosomes 13, 14, and 15. G7 and iG7 also display a 400-kb deletion that includes CDKN2A and CDKN2B (small arrow). (D,E) PCA of global gene expression (D; see also Supplemental Fig. 2A) and hierarchical clustering (E) of the 50 most significantly differentially expressed genes for normal NS cells and GNS cells (CB660, G7, and G26), hESCs and two clonal GiPSCs (iG7-1, iG7-2; iG26-1, and iG26-2), and iPSCs (iCB660) confirms that iG7 and iG26 are extensively reprogrammed to an ESC-like state. Analyzed iPSCs and GiPSCs were between passages 6 and 11.

Figure 3.

GNS cells possess epigenetic anomalies common in GBM that can be reset following reprogramming of GNS cells. (A,B) Scatter plots depicting percentage of DNA methylation changes identified using Infinium Human Methylation27 BeadChip arrays (Illumina, Inc.). Each dot represents a distinct CpG site. cMVPs hypermethylated in both G7 and G26 appear in blue, and hypomethylated loci are in green. Normal NS cells (CB660) versus either G7 or G26 identify tumor-specific methylation changes on genes such as CDKN1C (p57KIP2) and TES, which were hypermethylated in both parental GNS lines (see also Supplemental Fig. 2). (C) Immunoblotting showing reduced expression of tumor suppressor genes TES (in all tested GNS lines) and CDKN1C (in several GNS lines, including G7 and G26) when compared with normal NS cells CB660 and CB152. (D) DNA methylation analysis of the OCT4 and NANOG promoters using pyrosequencing. An average methylation level of >50% is depicted as a black circle, while levels <50% are shown as white circles. GNS cells contain extensive DNA methylation at both promoters, and these are removed following reprogramming. Numbers are percentage of average DNA methylation at the OCT-4 and NANOG promoters. (E–H) Scatter plots depicting DNA methylation levels (percentage) analyzed with Infinium Human Methylation27 BeadChip arrays (Illumina, Inc.). Each dot represents a distinct CpG site. cMVPs hypermethylated in both G7 and G26 appear in blue, and hypomethylated cMVPs appear in green. (E,F) iG7-1(GiPSC) versus G7 (GNS) and iG26-1 (GiPSC) versus G26 (GNS) illustrate extensive changes in methylation patterns after reprogramming. (G,H) iG7-1 versus iG7-2 and iG26-1 versus iG26-2 illustrate similarities between individual GiPSC clonal lines. Analyzed GiPSC clones were between passages 6 and 11.

Figure 4.

GiPSCs form multilineage teratomas in vivo. (A) Appearance of teratoma-like tumors generated following transplantation of GiPSCs into the right kidney capsule (iG7 and iG26, P8–P11). (L) Left control kidney; (R) right control kidney. iG7 and iG26 tumors were similar in macroscopic appearance to normal iPSC-derived tumors (B) iG7 and iG26 gave rise to immature teratomas. Examples of immature neural-like rosettes (H&E; iG7, bottom panel; iG26, top panel) as well as more differentiated nonneural tissues such as mesenchymal cartilaginous differentiation (H&E; iG7, top panel) muscle (mesoderm; H&E; iG26, bottom panel), glandular structures (endoderm and CEA⁺) and nonneural ectoderm (hair follicle CAM5.2⁺). Similar results were obtained for tumors derived from both kidney capsule and subcutaneous injections. (Top panels) Brain transplantation of iG7 also gave rise to immature teratomas with regions of nonneural tissues. (C) Mitotic markers Ki67 and phospho-histone H3 (PPH3) are observed at higher frequency in GiPSC tumors than control iPSCs. (D) Teratomas contain CDKN1C- and TES-expressing tissues.

Figure 5.

Demethylated tumor suppressor genes can be transcriptionally activated after reprogramming. (A) Immunocytochemistry for the pluripotency markers NANOG and OCT4 before and after in vitro differentiation to EBs. iG7 and iG26 lines are immunopositive for NANOG and OCT4, while the parental GNS lines G7 and G26 are not. NANOG and OCT4 immunoreactivity was down-regulated following differentiation. (B,C) Immunostaining and immunoblotting, respectively, show activation of previously silenced tumor suppressor genes TES and CDKN1C during EB differentiation. (D,E) Ki67 immunocytochemistry was used to score proliferating cells in EB differentiations (10-d + 7-d serum-containing medium) of GiPSC lines and revealed a significant and consistent increase in numbers of cycling cells between GiPSCs and normal iPSCs (iCB660) (P = 8–14). (*) P < 0.03, Student's t-test. See also Supplemental Figure 5.

Figure 6.

GiPSC-derived neural progenitors remain malignant. Neural progenitors (N-iG7-1 and N-iG7-2) were generated by in vitro differentiation of GiPSCs (P12–P14) (A) and injected in the striatum of adult mice (B). Immunostaining (C), immunoblotting (D), and correlation analysis (E) of the expression of 189 NS cell markers using qRT–PCR on custom TLDAs. NS cells (CB660, CB541, and CB192), GNS cells (G7 and G26), GiPSCs (iG7-1 and iG7-2), and neural (N-iG7-1 and N-iG7-2) and nonneural progeny of GiPSCs (M-iG7-1 and M-iG72) show that neural differentiated GiPSC cultures express neural markers at levels comparable with G7. (F) Quantification and correlation of reprogrammed cMVPs. A majority (80% and 83%) of the normalized cMVPs persist in differentiating GiPSCs (N-iG7 and M-iG7). (G) Kaplan-Meier blot depicting the survival of a cohort of 18 adult mice that had 100,000 G7 or N-iG7 cells injected into the striatum. (H) Coronal sections of typical examples of forebrains from mice injected with 100,000 G7 or N-iG7-1 cells after 18 wk. (IS) Injection site; (CI) contralateral side. Immunohistochemistry for human Nestin (hNestin) and Ki67 indicates that N-iG7 cells are highly proliferative and disperse widely from the injection site. Similarly to G7, N-iG7 cells infiltrated the contralateral side of the brain in most cases.

Figure 7.

GiPSC-derived mesodermal progenitors are no longer infiltrative. (A) Nonneural mesodermal cells (M-iG7-1 and M-iG7-2) were generated by in vitro differentiation of GiPSCs (P12–P14). Immunocytochemistry for the neural marker BLBP and Nestin and the nonneural epithelial marker keratins 7 and 8 (CAM5.2) confirms that in contrast to G7, M-iG7 cells show no expression of NS cell markers but partially express nonneuronal epithelial keratins. (B) Top three gene ontology terms (sorted by odds ratio) for those genes specifically induced in M-iG7 cells after differentiation. (C,D,F) Coronal sections of typical examples of forebrains from mice injected with 100,000 G7 and M-iG7-1 cells after 18 wk. (IS) Injection site; (CI) contralateral side. M-iG7-1 formed benign, noninfiltrative tumors that lacked pluripotency marker (see Supplemental Fig. 6E), expressed TES, and stained positive for alcian blue (indicative of cartilage). This contrasted with the highly infiltrative behavior of G7. See also Figure 6. Ki-67-positive cells were present in the benign mass but rare. (E) Immunoblotting for tumor suppressors, pluripotency marker NANOG, and radial glia/NS cell marker BLBP. CDKN1C (F) and TES (D) are only detectable in nonneural cultures (M-iG7-1 and M-iG7-2) or tumors.