Altered Expression of Polycomb Group Genes in Glioblastoma Multiforme
Citation: Li G, Warden C, Zou Z, Neman J, Krueger JS, et al. (
Altered Expression of Polycomb Group Genes in Glioblastoma Multiforme
Gang Li 0
Charles Warden 0
Zhaoxia Zou 0
Josh Neman 0
Joseph S. Krueger 0
Alisha Jain 0
Rahul Jandial 0
Mike Chen 0
Helen Fillmore, University of Portsmouth, School of Pharmacy & Biomedical Sciences, United Kingdom
0 1 Division of Neurosurgery, Department of Surgery, City of Hope National Medical Center, Duarte, California, United States of America , 2 Bioinformatics Core , Department of Molecular Medicine, City of Hope National Medical Center, Duarte, California, United States of America , 3 Flagship Biosciences, Boulder, Colorado , United States of America
The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.
Funding: This study was supported by Margaret E. Early Medical Research Trust Award 2012 (to MC). The funder had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Manuscript co-author Mike Chen is a PLOS ONE Editorial Board member, and manuscript coauthor Joseph S. Krueger is currently an
employee of Flagship Biosciences, Boulder, Colorado 80301. These do not alter the authors adherence to all the PLOS ONE policies on sharing data and materials.
The authors have declared that no other competing interests exist.
Glioblastoma multiforme (GBM) is an incurable primary brain
tumor of the astrocytic lineage. Current therapies are modestly
effective. Even with the most sophisticated treatment, median
survival for patients with GBM is little more than a year . To
improve the ability to diagnose, treat, and prevent GBM, a better
understanding of the molecular basis of this disease is necessary.
Recently, epigenetic aberrations have been implicated in the
development of GBM [2,3]. For example, in GBMs cancer specific
concordant hyper-methylation of the CpG islands suppresses the
promoters of hundreds of genes, giving rise to the glioma-CpG
island methylator phenotype (G-CIMP) . In addition to DNA
methylation, another epigenetic mechanism involved in
carcinogenesis appears to be malfunction or dysregulation of proteins that
function in chromatin modification and remodeling. For example,
EZH2 and BMI-1, both of which are reported to be involved in
the maintenance and renewal of GBM stem cells, are also
overexpressed in GBMs [5,6]. EZH2 and BMI1 are members of
Polycomb group (PcG) proteins. PcG family members are
important epigenetic regulators that generally form protein
complexes to perform their functions. The two main polycomb
group complexes in mammals are Polycomb repressive complex 1
(PRC1), in which BMI-1 is a component and Polycomb repressive
complex 2 (PRC2) in which EZH2 is an enzymatic component.
PRC1 compacts chromatin and catalyses the monoubiquitination
of histone H2A while PRC2 catalyses the methylation of histone
H3 at lysine 27 to repress gene expression [7-9]. PcG mediated
chromatin modification exerts a major influence on the
maintaining of gene expression patterns of different cells that are set during
early development and differentiation.
Given the complex interaction that occurs between PcG
proteins, we hypothesized that large-scale dysregulation that is
not confined to aberrant expression of EZH2 and BMI1 was
needed for the development or maintenance of GBMs. To this
end, we systematically interrogated The Cancer Genome Atlas
(TCGA) database for expression of all of the PcG genes. In
addition to previously reported alterations in EZH2 expression, we
found remarkable differences in the expression of several PcG
genes between GBMs and normal brain tissue. Of particular
interest, our data revealed that expression of Chromobox 6
(CBX6), a component of PRC1, was suppressed in cancer
specimens. Once this finding was validated, we demonstrated that
CBX6 overexpression inhibited cell proliferation, suggesting that
aberrant expression of CBX6 potentially promotes the
development of GBMs.
Materials and Methods
Interrogation of The Cancer Genome Atlas (TCGA)
database. TCGA data portal was accessed at:
expression data from the Agilent G4502A_07 platform was used for
initial analysis. A gene was considered overexpressed if the log2
Tumor/Normal Ratio was greater or equal ($) to 0.5 or
downregulated if the log2 Tumor/Normal Ratio was less or equal
(#) to -0.5. When stringent criteria were applied, a gene was
considered overexpressed or downregulated if the log2 Tumor/
Normal Ratio$1 or#-1 respectively.
Differential Gene Expression. Three studies [10-12]
containing glioma samples of different grades and normal brain tissues
were used to calculate differentially expressed genes that are up or
down regulated in gliomas. P-values were calculated via t-test, and
False Discovery Rates (FDR) were calculated based upon the
distribution of t-test p-values using the method of Benjamini and
Hochberg . Genes were considered differentially expressed if
they showed a fold-change greater than 1.2 with a FDR,0.05.
Visualization of Gene Expression for TCGA
Subtypes. Robust multichip average (RMA) normalization
 was applied to raw expression TCGA data (.CEL files for
Affymetrix arrays) . Molecular subtype labels from Verhaak et
al.  were applied to the samples used in the training dataset for
that study (N = 177). Expression was averaged among probes for
genes with multiple probes on the Affymetrix HT HG-U133A
array. The Partek Genomics Suite (Version 6.5) was used for
normalization, hierarchical clustering, and dot-plots.
Overall survival status and overall survival time was used to
create survival plots for matched probes for PcG genes for 6
patient cohorts [10,11,15,1719]. Patients were divided into high
and low expression groups based on median gene expression.
Kaplan-Meier plot, log-rank test on the overall survival data was
performed on this combined dataset. The p-value was calculated
by log-rank test for the two-groups. 803 GBM patient samples
were included in the survival analysis.
Human primary astrocytes derived from human cerebral cortex
(Cell Applications Inc.) were cultured in Astrocyte Growth
Medium (Cell Applications Inc.). T98G and U251MG
glioblastoma multiforme cell lines were purchased from ATCC, and
cultured in Dulbeccos Modified Eagle Medium supplemented
with GlutaMAX and 10% fetal bovine serum (Hyclone).
Myc-DDK-tagged human CBX6 cDNA cloned into the
pCMV6-Entry vector was purchased from OriGene. The
pCMV6-Entry CBX6 construct was cut with EcoRI and
religated to serve as a control vector.
Generation of Tetracycline-inducible CBX6
Overexpressing Cell Lines
U251MG cells were first stably transfected with two constructs,
pGL4.51[luc2/CMV/Neo] (Promega) and pcDNATM6/TR (Life
Technologies), the clone with the highest combined luciferase and
Tet repressor expression was further stably transfected with a
pcDNATM4/TO-CBX6 construct, the resulting clones were
named as U251MG-Luc/TR-CBX6TO (For detail, please see
EZH2 (#5246S, Cell Signaling Technology) and GAPDH
(#5174S, Cell Signaling Technology).
Brain glioblastoma multiforme tissue arrays (GL806a) were
purchased from US Biomax Inc. Antigen retrieval was performed
in 0.01M citrate buffer (pH 6.0) in a microwave oven.
PictureTM 3rd Gen Immunohistochemistry (IHC) Detection Kit
(Invitrogen) was used for immunodetection of CBX6. The slides
were scanned on an Aperio ScanScope by Flagship Biosciences
(Westminster, CO.) and examined by two independent examiners.
IHC staining was quantified using the H-score which was
calculated by the formula: 3 X percentage of strongly staining
nuclei + 2 X percentage of moderately staining nuclei + 1 X
percentage of weakly staining nuclei, giving a range of 0 to 300.
Quantitative RT PCR (qRT-PCR)
Total RNA was isolated using the Trizol reagent (Invitrogen).
First-strand cDNA was synthesized using SuperScript III Reverse
Transcriptase (Invitrogen) and oligo(dT)20. Quantitative PCR was
performed in triplicate using SYBR green reagent (Bio-Rad) in the
iQ5 machine (Bio-Rad). At least three independent experiments
were performed for each assay. For CBX6, the sequences of the
forward and reverse primers were
59-AAACGGCGGATCCGAAAGGGAC-39 and 59-GCTGCAATGAGCCGCGAGTC-39
respectively. For GAPDH, the sequences of the forward and
reverse primers were 59-AGGTGAAGGTCGGAGTCAAC-39
Proteins were resolved using the Novex NuPAGE SDS-PAGE
Gel System (Invitrogen) with 3-(N-morpholino) propanesulfonic
acid (MOPS) running buffer and transferred to a nitrocellulose
membrane (Bio-Rad). The enhanced Chemiluminescent (ECL)
substrate from Pierce was used to detect horseradish peroxidase
(HRP) activity from HRP linked secondary antibody (Cell
Colony formation assay
pCMV6-Entry CBX6 and control constructs were transfected
into U251MG cells using LipofectamineH 2000 (Invitrogen), and
then cultured in growth medium containing selection drug G418
(Sigma-Aldrich) at 1 mg/ml for three weeks. The colonies were
fixed with 10% formalin, stained with 0.05% crystal violet
(SigmaAldrich) and counted manually. Alternative quantification was also
performed by measuring the absorbance at 540 nm after
solubilizing cell-bound crystal violet using pure methanol.
Statistical analysis was performed using GraphPad Prism
(GraphPad Software, La Jolla, CA). Differences between the
Hscores for CBX6 were analyzed by the MannWhitney U test.
qRT-PCR data and data of the colony formation assay were
analyzed by Students t-test. For all tests, a P value,0.05 was
considered to be significant.
mRNA Expression of PcG genes in GBM
PcG gene products were initially identified as transcriptional
repressors that when mutated resulted in dramatic changes of
body patterning in Drosophila . In mammals, PcG proteins
form two major protein complexes, PRC1 and PRC2, to execute
profiling, several subtypes of GBM have recently been identified.
Verhaak et al. classified glioblastoma samples into four subtypes,
i.e., classical, proneural, neural and mesenchymal subtypes using
unsupervised hierarchical clustering analysis . We examined
the TCGA dataset to determine if PcG genes were differentially
expressed in these GBM subtypes [15,16]. Pair-wise comparisons
revealed that the majority of the PcG genes were not differentially
expressed in different subtypes of GBM (Table S2). Exceptions
occurred with HDAC2 and JARID2, which were significantly
upregulated in the proneural subtype, and with EZH2, which also
had higher expression in proneural subtype but only when
compared to the mesenchymal subtype (Figure 2A).
their functions. However, polycomb mediated gene silencing in
mammals is complicated because there are usually multiple
orthologs for each PcG protein (Table S1). Hence, to determine
the role of PcGs in GBMs, it was necessary to comprehensively
analyze TCGA data for aberrant expression of all of the PRC1
and PRC2 genes. We also examined several PRC2 associated
genes such as DNMT3A, DNMT3B, SIRT1 and HDAC2.
Stringent criteria were used to determine relative expression of a
gene in GBMs (Tumor) to normal brain (Normal). Overexpression
occurred when the Tumor/Normal Ratio$2, whereas
downregulation was indicated by a Tumor/Normal Ratio#0.5. As shown
in Figure 1, EZH2 overexpression was present in 98.6% of GBM
samples, which is consistent with previous reports [6,21]. Other
PcG genes commonly overexpressed in GBMs include PHF19
(61.8%), CBX8 (55.4%) and PHC2 (51.2%). Contrary to previous
studies [5,22], overexpression of BMI1 in GBMs was not observed.
PcG genes that were downregulated included CBX6 (82.5%),
CBX7 (96.9%), RYBP (49.3%) and EZH1 (48.6%). We also
performed less stringent analysis using Tumor/Normal Ratio$1.4
and Tumor/Normal Ratio#0.7 as criteria for overexpression and
downregulation respectively. The results, which follow the same
trend as the results of the high stringent analysis are provided as
supplementary data (Figure S1).
To validate the dysregulation of these PcG genes in GBMs, we
performed further analysis using three independent published data
sets . Consistent with the TCGA analysis, EZH2 was
found to be significantly upregulated (6.4 to 12.9 fold changes) in
GBMs samples of all three datasets. PHF19 and PHC2 were also
up-regulated in the three datasets, whereas CBX8 was found to be
upregulated in only one of the independent datasets. Likewise, it
also revealed the consistent downregulation of CBX6, CBX7,
RYBP and EZH1 in GBMs samples of all three datasets (Table 1
& 2). The confirmation of PcG dysregulation in independent
datasets indicates the observed changes in PcG gene expression is
not due to artifacts introduced by numerous confounding factors.
PcG expression as a function of GBM subtype
While all GBMs share similar histological characteristics, e.g.,
presence of anaplastic glial cells, mitotic activity, vascular
proliferation and necrosis, there is significant heterogeneity in
the molecular profiles of GBMs amongst patients or even within
an individual patients tumor [23,24]. Based on gene expression
Figure 1. Interrogation of The Cancer Genome Atlas (TCGA) database of mRNA expression of PcG genes in glioblastoma
multiforme (GBM). PcG gene expression statuses in glioblastoma patients were allocated into 3 different categories, overexpression (Tumor/
Normal Ratio$2, red); downregulation (Tumor/Normal Ratio#0.5, green), and no change (0.5#Tumor/Normal Ratio#2, yellow). The stacked bar
graph depicts the percentage of each category, calculated out of total 424 patients.
Figure 2. Expression of PcG genes in different glioblastoma subtypes and astrocytoma grades. (A) PcG gene expression in TCGA
glioblastoma multiforme (GBM) subtypes. Using the TCGA data for GBM, pair-wise comparisons were conducted between all 4 of the molecular
subtypes (Mesenchymal, Classical, Neural and Proneural, color coded). ** P,0.01. (B) Continuum of abnormal expression in PcG genes in different
histological grades of astrocytomas. The Y-axis measures the background adjusted Robust Multi-array Average (RMA) which indicates the normalized
expression level. Box-and-whisker plots show the distribution of mRNA expression in normal brain and different grades of astrocytomas (color coded).
For detailed statistical analyses, please see Table S3 & S4.
Expression of PcG genes in different astrocytoma grades
GBMs (WHO Grade IV) are the most malignant form of
astrocytoma. We speculated that there would be a continuum of
PcG dysregulation as the astrocytoma became progressively more
malignant. PcG gene expression was then examined in
astrocytoma samples with different histological grades. Interrogation of a
combined dataset  revealed that a grade-wise progression
of PcG dysregulation was evident for select genes. CBX6, CBX7
and EZH1 shared a similar pattern and correlated negatively with
increasing astrocytoma grade, whereas the opposite occurred with
EZH2 and PHF19 (Figure 2B, table S3 and S4).
Correlation between PcG expression and overall survival
of GBM patients
Next, we examined a combined dataset including 6 GBM
patient cohorts [10,11,15,1719] to determine if the PcG genes
that were the most differentially expressed were related to GBM
patient survival. We rationalized that genes with aberrant
expression in GBM that also correlated with survival would be
attractive candidates as therapeutic targets and/or biomarkers.
Unfortunately, Kaplan-Meier survival analyses revealed that none
of the dysregulated PcG gene significantly correlated with GBM
patient survival (data not shown). However, in a cohort which
includes low grade gliomas , significant survival benefits were
observed for patients with low EZH2, PHF19, CBX8 and PHC2
expression, and high CBX7, CBX6, RYBP and EZH1 expression
(Figure S2). Nonetheless, this benefit might only manifest those
genes are differentially expressed between low and high grade
Expression of CBX6 is downregulated in GBM cell lines
Out of the list of PcG genes that were analyzed in greater depth,
we were most intrigued by the changes of the five Chromobox
homolog genes (CBXs), which are the mammalian orthologs of
Drosophila Polycomb (PC) gene. Through the chromodomain of
CBX proteins, PRC1 complexes bind to the trimethylated lysine
27 of histone H3 (3mH3K27), the product of PRC2 catalysis, to
find gene targets. As shown in Figure 1, the expression levels of
CBX6 and CBX7 are downregulated, and the expression of
CBX8 is upregulated in glioblastoma samples, whereas the
expression of CBX2 and CBX4 remain unchanged. Cell lines
provide a homogeneous population, and a strong positive
correlation has been observed in gene expression patterns between
cancer cell lines and primary tumors . To extend the above
observation, the expression of CBX6, CBX7, CBX8 and EZH2 in
primary human astrocytes were compared with their expression in
two GBM cell lines, T98G and U251MG. As shown in Figure 3,
compared to their levels in primary astrocytes, expression of
CBX6 is significantly downregulated, whereas the expression of
CBX8 and EZH2 are upregulated at both the mRNA and protein
levels in the GBM cell lines. On the other hand, CBX7 expression
level was down-regulated in T98G, but not in U251MG cells
glioblastoma cells, when compared to levels in primary astrocytes.
Because of that CBX7 mRNA level is extremely low in normal
primary astrocytes, approximately 20 times lower than the level of
CBX6 (data not shown), we chose CBX6 for further studies.
Expression of CBX6 is downregulated in clinical glioma
Next we analyzed CBX6 levels by immunohistochemistry using
a tissue microarray containing 29 GBM cases and 5 samples of
normal brain tissue (Figure 4A). To objectively describe the extent
of CBX6 immunohistochemical staining, the degree of staining
was quantified using the H score method (Figure 4B). All 5 samples
of normal brain tissue were positively stained for CBX6 with a
median H score of 83.2. Among the 29 GBM samples, 22 samples
showed weak or no CBX6 staining with H score below 5, 5
samples showed modest staining with H score between 5 and 40
and 2 samples showed comparable staining to normal brain tissue
(Figure 4B). These results confirmed that CBX6 expression is
indeed downregulated in GBM samples, although some
Overexpression of CBX6 leads to cell growth arrest
To determine if CBX6 dysregulation has any functional
ramifications as opposed to merely being a passenger alteration,
we examined the effect of CBX6 overexpression on GBM cell
proliferative capacity using a colony formation assay. U251MG
cells were transfected with a construct encoding the CBX6 cDNA
and a neo(R) cassette. After 21 days of G418 selection, the cells
transfected with CBX6 formed significantly fewer and smaller
colonies compared to the vector control (Figure S3A). Similar
results were also obtained when using T98G glioblastoma cells
(Figure S3B) indicating that CBX6 has an inhibitory effect on cell
growth. To provide a well-controlled system to dissect the CBX6
function; we then established multiple tetracycline-inducible
CBX6 overexpression stable cell lines based on U251MG
(Figure 5A), and examined the effect of CBX6 overexpression
on proliferative capacity using colony formation and ATP assays.
U251MG-Luc/TR-CBX6TO cells (line 17) were treated with
doxycycline (+dox) to induce CBX6 overexpression. Compared to
non-induced controls, the cells with induced CBX6 overexpression
formed fewer colonies (Figure 5B). The cell proliferation inhibitory
effect was also corroborated by measuring the absorbance after
solubilizing cell-bound crystal violet (Figure 5C). Further, levels of
ATP of U251MG-Luc/TR-CBX6TO (+dox) cells (line 6 and 17)
were dramatically decreased at days 3 and 4 compared to
U251MG-Luc/TR-VectorTO (vector control, +dox) cells,
indicating cell proliferation was reduced upon overexpression of CBX6
(Figure 5D). The later control was included to isolate CBX6 effects
from the potentially confounding effect of doxycycline on cell
proliferation. Collectively, these data suggest that CBX6 has an
inhibitory effect on cell growth in vitro.
PcG proteins have been shown to play a significant role in the
epigenetic maintenance of cell identity. Unsurprisingly, recent
studies strongly suggest that dysregulation of PcG proteins can
influence the development or malignant evolution of cancers [26
28]. Indeed, PcG proteins EZH2, BMI1 and CBX7 have been
shown to possess oncogenic or tumor suppressor functions in
different tumors including GBMs [5,6]. Compared with
Drosophila, PcGs evolved towards greater complexity in mammals with
approximately 28 family members . Because many PcGs
have orthologs that can substitute for each other in the functional
complexes such as PRC1 and PRC2, we speculated that global
imbalances maybe present because certain components were
already known to be dysregulated.
A systematic bioinformatics analysis of PcG expression in GBMs
was conducted using the TCGA database in this study. The survey
revealed that EZH2, PHF19, CBX8 and PHC2 were the most
frequently upregulated PcG genes; and CBX7, CBX6, EZH1 and
RYBP were the most frequently downregulated PcG genes. The
most frequently overexpressed PcG gene in GBMs was EZH2 (two
fold increase in 98.6% GBMs). This finding is in agreement with
previous reports [6,21] and also consistent with the observed
overexpression of EZH2 in many different types of solid tumors
[29,30]. EZH2 appears to be a promising therapeutic target in
GBMs because of the extremely high frequency of overexpression
and the observations that EZH2 inhibition severely retards the
growth of cancer cells [6,21].
Another discovery was that multiple Chromobox (CBX) genes
had aberrant expression in GBMs. CBX proteins are mammalian
homologues of the drosophila Polycomb (Pc) protein and
components of mammalian PRC1 complexes. Mammalian
genomes encode five homologs of drosophila Polycomb (Pc) gene:
CBX2, CBX4, CBX6, CBX7 and CBX8. Binding of CBXs
through their chromodomain to H3K27me is the classical model
for PRC1 recruitment , although there are evidences
supporting that H3K27me3/CBX-independent mechanisms can also
recruit PRC1 complexes . Though poorly understood, it
has been suggested that each CBX protein has different functions
. Indeed, compared to normal brain, the expression of
CBX2 and CBX4 does not change, CBX6 and CBX7 decreases,
while the expression of CBX8 increases in GBMs indicating the
five CBXs are differentially regulated. Both CBX7 and CBX8
have been reported to repress the INK4a/ARF tumor suppressor
locus allowing normal cells to bypass senescence [39,40]. Studies
have not shown expression changes of CBX6 or CBX8 in tumors,
whereas there are contradictory reports on changes of CBX7
expression. For example, CBX7 expression is upregulated in
lymphomas and gastric tumors [41,42], but downregulated in
malignancies such as colon, thyroid, pancreatic, and urothelial
carcinomas . The discovery of CBX6 downregulation in
GBMs is particularly intriguing, because virtually nothing is
known about CBX6, and downregulation of CBX6 is marked and
occurs consistently in independent GBM samples. Our data also
showed that overexpression of CBX6 led to cell growth arrest,
further studies are needed to address the mechanism behind this.
Understanding the functional significance of the reciprocal
expression pattern of CBX8 relative to CBX6 and CBX7 in
GBMs might eventually leads to the development of compounds
targeting the chromodomain of a specific CBX protein which
potentially have anti-tumor efficacy.
Dozens of studies have used gene expression profiling to subtype
gliomas to complement the morphological classification of gliomas
. GBM subtyping based on gene expression profiling
represents a significant step forward towards the development of
personalized treatment being tailored to the unique pattern of
genetic changes in each patients tumor. In this study we asked
whether PcG genes are differentially expressed among the
different subtypes of glioblastoma defined by TCGA . The
analysis revealed that the majority of the PcG genes are not
differentially expressed in different subtypes of GBM. On the other
hand, our study revealed that the histological grade of
astrocytomas correlated with the degree of aberrant expression in a graded
manner in many PcG genes. Among them, the expressions of
EZH2 and PHF19 correlated positively with the astrocytoma
grades, whereas the expressions of CBX7, CBX6 and EZH1
correlated negatively with astrocytoma grades. One interpretation
of these findings was that these changes could occur due to active
cell division since higher grades have greater proliferative capacity.
Another, more intriguing possibility was that PcG expression
changes were a cause or essential event for malignant progression.
In summary, emerging evidence indicates glioblastomas adopt
changed epigenetic regulating machinery to maintain its
malignancy as manifested here by altered expression of PcG proteins. In
this study, we demonstrate that another member of the PcG
family, CBX6, has a potential role in gliomagenesis. Specifically,
we showed that expression of CBX6 is downregulated in
glioblastoma cell lines and clinical samples and that induced
CBX6 overexpression inhibited glioblastoma cell proliferation.
Future studies will be required to assess its functional significance,
elucidate its targets in the astrocytic genome, to corroborate the
above findings using other in vitro and in vivo GBM model
systems, and to understand why glioblastoma utilizes different
CBXs during its malignant progression.
Figure S1 Interrogation of The Cancer Genome Atlas
(TCGA) database of mRNA expression of PcG genes in
glioblastoma multiforme (GBM). PcG gene expression
statuses in glioblastoma patients were allocated into 3 different
categories, overexpression (Tumor/Normal Ratio$1.4, red);
downregulation (Tumor/Normal Ratio#0.7, green), and no
change (0.7#Tumor/Normal Ratio#1.4, yellow). The stacked
bar graph depicts the percentage of each category, calculated out
of total 424 patients.
Figure S2 Kaplan-Meier survival estimates overall
survival of glioma patients according to the PcG
expression. A risk score was assigned to each patient which is a
linear combination of the expression levels of the dysregulated
PcG genes weighted by their respective upregulation or
downregulation status. Specifically, the risk scores are calculated as follows:
Risk score = EZH2 + PHF19 + CBX8 + PHC2 - CBX7 - CBX6
RYBP - EZH1. Patients are divided into two groups based on
median expression, and the Kaplan-Meier method was used to
estimate overall survival time for the two groups. Statistical
significance was analyzed using the two-sided log rank test.
Median survival time for patients with high risk score (n = 135) is
10.5 months, whereas median survival time for patients with low
risk score (n = 131) is 35.2 months, p = 3.09e-08.
Figure S3 Overexpressing CBX6 gene inhibits the
growth of glioblastoma cells. (A) & (B) U251MG (A) or
T98G (B) cells were transfected with a vector expressing CBX6
cDNA, then put under drug (G418) selection for 21 days. The
colonies were stained with 0.05% crystal violet. The empty vector
pCMV6-Entry was used as a control. Shown is a representative of
two independent experiments. (C) Number of colonies of
U251MG cells were counted and graphed. Error bars represent
standard deviation. * (P,0.05). Top panel, western blot analysis
shows CBX6 is overexpressed in the transfected cells. (D)
Methanol was added to solubilize the crystal violet dye.
Absorbance at 540 nm was read using DTX 880 plate reader.
Error bars represent standard deviation. * P,0.05. Top panel,
western blot analysis shows CBX6 is overexpressed in the
Figure S4 Diagrams of the vectors transfected into
U251MG glioblastoma cell lines. CMV, CMV promoter;
Firefly (luc2) encodes firefly luciferase gene luc2; TetR, encodes
Tet repressor gene; 2 x TetO2, two copies of the tet operator 2
(TetO2) sequence; Neor, Blastr and Zeocinr represent Neomycin,
Blasticidin and Zeocin resistance gene cassettes respectively.
List of Polycomb Group Proteins
We thank Dr. Massimo DApuzzo for help in histologic examination, Drs.
Jun Wu and Leying Zhang for helpful discussions. We are also extremely
grateful for the generous funding for this research provided by Mrs. Janis
Wechter in honor of her husband Larry Wechter.
Conceived and designed the experiments: GL MC. Performed the
experiments: GL CW ZZ AJ. Analyzed the data: GL CW JSK RJ MC.
Contributed reagents/materials/analysis tools: JN JSK. Wrote the paper:
GL CW MC.
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