TRIM11 is overexpressed in high-grade gliomas and promotes proliferation, invasion, migration and glial tumor growth
TRIM11 is overexpressed in high-grade gliomas and promotes proliferation, invasion, migration and glial tumor growth
TRIM11 (tripartite motif-containing protein 11), an E3 ubiquitin ligase, is known to be involved in the development of the central nervous system. However, very little is known regarding the role of TRIM11 in cancer biology. Here, we examined the expression profile of TRIM11, along with two stem cell markers CD133 and nestin, in multiple glioma patient specimens, glioma primary cultures derived from tumors taken at surgery and normal neural stem/progenitor cells (NSCs). The oncogenic function of TRIM11 in glioma biology was investigated by knockdown and/or overexpression in vitro and in vivo experiments. Our results showed that TRIM11 expression levels were upregulated in malignant glioma specimens and in high-grade glioma-derived primary cultures, whereas remaining low in glioblastoma multiforme (GBM) stable cell lines, low-grade glioma-derived primary cultures and NSCs. The expression pattern of TRIM11 strongly correlated with that of CD133 and nestin and differentiation status of malignant glioma cells. Knock down of TRIM11 inhibited proliferation, migration and invasion of GBM cells, significantly decreased epidermal growth factor receptor (EGFR) levels and mitogen-activated protein kinase activity, and downregulated HB-EGF (heparin-binding EGF-like growth factor) mRNA levels. Meanwhile, TRIM11 overexpression promoted a stem-like phenotype in vitro (tumorsphere formation) and enhanced glial tumor growth in immunocompromised mice. These findings suggest that TRIM11 might be an indicator of glioma malignancy and has an oncogenic function mediated through the EGFR signaling pathway. TRIM11 overexpression potentially leads to a more aggressive glioma phenotype, along with increased malignant tumor growth and poor survival. Taken together, clarification of the biological function of TRIM11 and pathways it affects may provide novel therapeutic strategies for treating malignant glioma patients.
TRIM11; oncogene; EGFR; malignant glioma; tumor formation
Malignant gliomas are the most common primary tumors in the
central nervous system and are characterized by rapid cell
proliferation, high invasiveness, genetic alteration and increased
angiogenesis.1 Despite continued advances in surgical and
medical therapeutics, the prognosis for patients diagnosed with
the most common and aggressive malignant glioma?
glioblastoma multiforme (GBM)?remains poor, with a median
survival of only 12?15 months.2
In recent years, glioma molecular markers such as MGMT
(O6methylguanine-DNA methyltransferase), EGFR (epidermal growth
factor receptor) and IDH1 (isocitrate dehydrogenase-1) have had
important roles in profiling the diagnosis, prognosis and disease
management in subtypes of glioma patients.2,3 More recently,
CD133 (prominin-1) and nestin have been considered as
prognosis markers for glioma patients as their expression is
associated with poor prognosis and correlates better with clinical
course than the histological grading.4,5 They are also used as
markers for glioma stem-like cells (GSCs), a small subset of tumor
cells situated at the apex of the cellular differentiation hierarchy
and responsible for sustaining long-term tumor growth through
self-renewal and the production of more differentiated daughter
cells.6?8 GSCs are known to contribute to tumorigenesis and
radiation resistance in malignant glioma9 and are regulated via
many signaling pathways, including Notch, Hedgehog and MAPK
(mitogen-activated protein kinase) cascades and so on.10 Due to
the malignancy-driving roles of GSCs, drugs that specifically target
the GSCs have a very important potential role in glioma therapy.11
However, both CD133 and nestin are also present on
nonmalignant neural stem/progenitor cells (NSCs),12,13 and targeting
GSCs through these markers will likely damage NSCs. This is
particularly problematic in the brain, as damage to NSCs resulting
from glioma therapies14 can produce profound cognitive side
effects that impair patient?s functional independence and quality
The tripartite motif-containing (TRIM) family is characterized by
unique structural motifs: a RING finger domain, a B-box domain
and a coiled-coil domain, making them members of the RING
B-box coiled-coil (RBCC) protein family.17?19 Members of this
family have been implicated in various cellular processes,
including development, neurodegenerative diseases, cellular
response to viral infection and cancer.20 As a typical RBCC
protein, TRIM11 functions as an E3 ubiquitin ligase and binds to
and destabilizes Humanin, a neuroprotective peptide against
Alzheimer?s disease-relevant insults.21 In addition, TRIM11
interacts with and destabilizes the activator-mediated cofactor
complex (ARC105), which in turn suppresses ARC105-mediated
transcriptional activation induced by transforming growth factor b
signaling.22 TRIM11 also binds to and mediates the ubiquitination
of PAX6 (paired box 6), a member of the PAX family of
transcription factors and has a key role in organogenesis of the
eye, pancreas and brain.23 Finally, TRIM11 is involved in the
specification of noradrenergic phenotype by interacting with
Phox2b, a homeodomain transcription factor that could modulate
development of noradrenergic neurons.24 These findings
implicate an important, albeit still incompletely defined, role of
TRIM11 in the function and development of nervous system.
Nonetheless, an oncogenic function of TRIM11 has yet to be
identified in cancer cells of glial origin that shares a lot of common
characteristics with the normal glial cells of the brain.25 In this
study, we not only provide evidence that TRIM11 expression levels
correlate with the malignancy of glioma but also investigate the
oncogenic function of TRIM11 in vitro and in vivo.
TRIM11 is over-expressed in human high-grade glioma tumors and
glioma-derived GSCs but not in NSCs, low-grade glioma-derived
GSCs or stable malignant glioma cell lines
To investigate the correlation between TRIM11 and malignancy of
glioma, immunohistochemical analysis of TRIM11 was performed
using patient-derived glioma samples (Figure 1a). Strong TRIM11
staining was detected in GBM tissues but much less in the Grade II
and normal brain tissues (representative results shown). Next, we
profiled TRIM11 mRNA levels using 19 diverse histological primary
GSCs derived from glioma patients (Table 1), four stable GBM cell
lines (U-251, D-54, LN229 and U-87) and three NSCs (SC23, SC27
and SC30). Overall, TRIM11 was low in all the GBM cell lines, 6 of 7
(85.7%) low-grade GSCs and all NSCs (normalized fold expression
o2.5). By comparison, 1 of 3 (33.3%) Grade III-GSCs and 7 of 9
(78%) GBM-GSCs had higher levels of TRIM11 (normalized fold
expression 42.5; Table 1 and Supplementary Figure S1a). The
expression pattern of TRIM11 in cells was further confirmed at the
protein levels. Western blotting data showed that TRIM11
expressed higher in GBM-GSCs (Supplementary Figure S1b),
consistent with its mRNA levels. To evaluate the role of TRIM11
on clinical prognosis, we compared the outcome of patients with
different expression levels of TRIM11. As shown in Table 1 (survival
data), the high-grade glioma patients with lower TRIM11 had a
better prognosis (average 13.5-month survival for GBM patients)
compared with patients with higher TRIM11 (average 7.3-month
survival for GBM patients).
Next, we compared TRIM11 mRNA levels to those of CD133.
Consistent with a previous report,26 the three NSC cultures
displayed excessively high CD133 (up to 3000-fold normalized
expression). Although all stable GBM cell lines and low-grade
glioma-derived GSCs (except DB51) showed very low CD133, CD133
was strongly expressed in all GBM-GSCs, and in one Grade III GSC
Abbreviations: GBM, glioblastoma multiforme; MG, malignant glioma; TRIM 11, tripartite motif-containing protein 11. aSurvival from surgery to death. bDead
unrelated to tumor progression.
(Table 1 and Supplementary Figure S1a). Our results were
consistent with previous reports4,5 that high CD133 mRNA levels
correlate with glioma malignancy. More importantly, GSCs with
elevated TRIM11 expressed concurrently higher CD133 (shading in
Table 1). For example, the two GBM-GSCs (DB32 and HuTuP01) that
showed the highest levels of TRIM11 (29.25 and 13.78-fold,
respectively) also displayed the highest levels of CD133 (66.07
and 27.34-fold, respectively), indicating a positive correlation
between TRIM11 and CD133 in gliomas. Next, we performed
immunofluorescent double staining to further compare the
expression parallel between CD133 and TRIM11 using
corresponding tissue sections. As shown in Figure 1b, both DB44 (Grade II) and
DB32 (GBM) revealed co-expression of TRIM11 and CD133, but the
strong immunopositivity was detected only in DB32. HuTuP01-GSC
was used to confirm these data. As shown in Figure 1c, TRIM11 was
not only localized diffusely mainly in the cytoplasm but also in the
nucleoplasm (top), which was consistent with previous reports,17,22
and strongly expressed on tumorsphere (bottom). Meanwhile, the
cells positive for TRIM11 staining also displayed strong CD133
immunopositivity in both tumorsphere and monolayer cells.
For nestin expression study, U-251 was used as a positive
control as previously described.27 Our results showed that nestin
expressed at elevated levels in almost all the glioma-derived GSCs
tested and more robustly in all GBM-GSCs and NSCs (Table 1 and
Supplementary Figure S1a).
Taken together, our results suggest that the levels of TRIM11
correlate with the malignancy of glioma and potentially with the
prognosis of glioma patients. Compared with CD133 and nestin,
TRIM11 might be a more specific indicator for GSCs as it is
upregulated only in malignant glioma-derived GSCs but not in
NSCs or low-grade glioma-derived GSCs.
TRIM11 mRNA levels correlate with differentiation status and
CD133 mRNA levels in gliomas
When cultured in serum-free medium, which allows for the
maintenance of an undifferentiated stem cell state, glioma cells
form tumorspheres, along with an increased expression of
CD133.28,29 To further investigate the correlation between
TRIM11, differentiation status and CD133 expression levels in
gliomas, we used serum-free stem cell medium (SCM) to culture
the stable glioma cell line D-54 and measured the changes in
TRIM11 and CD133 mRNA levels by quantitative
reversetranscriptase?PCR. When cultured in 10% fetal bovine
serumcontaining medium (differentiation medium), D-54 grew as a
monolayer and attached to the culture flask (Supplementary
Figure S2a, left photo). After re-seeding in SCM, they grew as
typical tumorspheres varied in size and floating in the medium
(Supplementary Figure S2a, right photo). In addition, both TRIM11
and CD133 were increased when cells were cultured in SCM
(Figure 2a), suggesting that D-54 cells cultured in stem cell
conditions can achieve the properties classically ascribed to GSCs
and confirming again a positive correlation between TRIM11 and
CD133. Next, using two GBM-GSCs that expressed highest TRIM11
and CD133 (DB32-GSC and HuTuP01-GSC), we switched culture
media from SCM to differentiation medium to induce GSC
differentiation. Accompanying with morphological changes
(Supplementary Figures S2b and c), TRIM11 and CD133
significantly decreased when GSCs underwent differentiation (Figures
2b and c (left panel)). Furthermore, when HuTuP01-GSC was
switched back to SCM (leading to a less differentiated state), both
TRIM11 and CD133 increased again (Figure 2c, right panel), and the
morphology of the cells changed back to that of the cells cultured
directly in SCM (Supplementary Figure S2c). By comparing with
early and late passages of DB32-GSC and HuTuP01-GSC, we found
both TRIM11 and CD133 decreased after continuous expansion
(Figure 2d), further confirming that TRIM11 levels are correlated
with differentiation status and CD133 levels of GSCs. The stem cell
character of HuTuP01-GSC was further identified by differentiation assay
(Supplementary Figure S2d), confirming their multipotential nature.
Downregulation of TRIM11 suppresses proliferation, migration and
invasion of glioma cells and inhibits EGFR expression, MAPK
signaling pathway and transcription of HB-EGF and CCND1
To investigate the potential oncogenic function of TRIM11 in
glioma, we knocked down TRIM11 in glioma cells through RNA
interference. Both protein and mRNA levels of TRIM11 were
decreased in siTRIM11 transfectants (Supplementary Figures 3a
and b). We next investigated the effect of TRIM11 on cell
proliferation using D-54. MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assay (Figure 3a, upper panel),
and bromodeoxyuridine (BrdU) cell proliferation analysis
(Figure 3a, lower panel) indicated that loss of TRIM11 was able
to significantly inhibit cell proliferation. Cell cycle analysis further
showed that downregulation of TRIM11 led to increased G1 phase
cells and decreased S phase cells (Supplementary Figure S3c).
The invasive behavior of malignant gliomas limits the
effectiveness of local therapies and contributes to their poor
prognosis.30 Therefore, wound closure assay (Figure 3b) was
performed to compare the migration capability between controls
and TRIM11 knockdown U-251 cells. TRIM11-downregulating cells
displayed a slower migration capability than control: while control
cells were able to migrate in and fill up the gap in 30 h, the gap
was only partially filled in siTRIM11 transfectants at the same time.
Similar results were found in LN229 cells (Supplementary Figure
S4a). The role of TRIM11 on cell invasion was next measured using
the Matrigel invasion chamber assay (Figure 3c). In both the D-54
and U-251 cells, suppression of TRIM11 inhibited cell invasion, and
the difference was statistically significant (Po0.05).
In GBMs, amplification and/or mutation of the gene encoding
EGFR occurs in up to 60% of tumors, and the constitutive
activation of EGFR can promote glioma cell proliferation and
invasion.31 As one of the main intracellular protein cascades of
EGFR, deregulated MAPK pathway due to activation of EGFR will
influence a diverse array of vital cellular functions, such as
proliferation, survival and differentiation.32,33 We next evaluated
the expression levels of EGFR and phosphorylation status of MAPK
in glioma cells having different TRIM11 levels. As shown in
Figure 3d and Supplementary Figure S4b, downregulation of
TRIM11 led to decreased expression of EGFR, p-c-Raf, p-MEK1/2
(MAPK/ERK kinases 1/2), and p-44/42MAPK, suggesting that
TRIM11 might regulate the EGFR/MAPK pathway. Meanwhile,
PI3K-AKT signaling, another downstream of EGFR, was not
affected by TRIM11 (Supplementary Figure S4c).
Next, we used the human EGF/PDGF (epidermal growth factor/
plateletderived growth factor) signaling PCR array to study
whether TRIM11 differentially regulate the transcription of any
Fold change (X2) (siCon vs siTRIM11)
Abbreviations: EGF, epidermal growth factor; PDGF, platelet-derived growth factor; TNF, tumor-necrosis factor; TRIM 11, tripartite motif-containing protein 11.
Note: Human EGF/PDGF Signaling PCR Array was performed to compare gene expression changes by TRIM11 downregulation. Genes with 4two-fold
regulation were considered as significant difference. The shading indicates the downregulation with > two-fold change.
genes in the EGF/PDGF signaling pathway. Table 2 showed the
profile of various differentially expressed genes with fold changes
of X2. There were six upregulated and four downregulated genes
in D-54 siTRIM11 (si-4) transfections, and one upregulated and four
downregulated genes in U-251 siTRIM11 (si-4) transfections.
Among all the genes changed, downregulation of two genes,
HB-EGF (heparin-binding EGF-like growth factor) and CCND1
(Cyclin D1), was found in both the cell lines. Meanwhile, the
expression of EGFR was not significantly changed. To confirm
these results, we measured again mRNA levels of EGFR, HB-EGF
and CCND1 using different primers (Figure 3e). Our results showed
that both HB-EGF and CCND1 were dramatically decreased, while
there was no statistically difference on EGFR in siTRIM11
transfectants. Similar results were found in LN229 cells transfected
with three different siTRIM11s (Supplementary Figure S4d).
To compare with knockdown experiments, gain-of-function
studies were further performed. Following transfection of a
FlagTrim11 construct (Supplementary Figure S5a), both the levels of
EGFR and the activity of MAPK pathway were increased
(Supplementary Figure S5b), further confirming an oncogenic
function of TRIM11 through promoting the accumulation of EGFR
and activity of MAPK cascade.
Overexpression of TRIM11 promotes tumorsphere formation and
proliferation in vitro and tumor growth in vivo
To further test the oncogenic function of TRIM11 both in vitro and
in vivo, transient and stable TRIM11 overexpressing transfectants
(pDsRed2-Trim11) were generated using mouse GL261 glioma
cells. Expression of TRIM11 mRNA was analyzed in Figure 4a
(stable transfection) and Supplementary Figure S5c (transient
transfection). Consistent with our TRIM11 knockdown results,
increased HB-EGF mRNA revealed in both transient
(Supplementary Figure S5c) and stable (Supplementary Figure
S5d) TRIM11 overexpressing cells. Interestingly, large
tumorspheres were detected in stable pDsRed2-Trim11 cells but not in
control cells (Figure 4a, right). This morphological change supports
our hypothesis that TRIM11 has an important role in achieving/
maintaining the stem cell state and led us to test the sphere
formation capability induced by upregulation of TRIM11. As shown
in Figure 4b, overexpression of TRIM11 significantly promoted
tumorsphere formation (upper panel), and the size of spheres
driven from pDsRed2-Trim11 cells was significantly larger than
that of control cells (right). Meanwhile, cell proliferation rate was
dramatically increased in pDsRed2-Trim11 cells when cells grew
on Matrigel-coated surface (Figure 4b, lower panel).
Based on our in vitro data (which suggest that TRIM11
significantly increases glioma cell proliferation and invasion), we
then tested whether TRIM11 overexpression can promote glioma
growth in vivo. Stable pDsRed2-Trim11-GL261 cells and control
cells were injected subcutaneous into BALB/C nu/nu mice. The
tumors generated by the pDsRed2-Trim11-GL261 cells were more
than three times larger (both tumor volume and weight) than the
tumors generated by the control cells (Figures 4c and d).
Furthermore, macroscopic examination of these tumors showed
increased vascularization, and extensive areas of necrosis
characteristics well described to predict an aggressive phenotype in
glial tumors. These findings further support that TRIM11
overexpression enhances glioma cell ability to form aggressive tumors
and correlate with our findings in human specimens, where high
TRIM11 levels correlated with poor prognosis.
Significant progress has been made on investigating the cellular,
molecular, and genetic changes involved in malignant gliomas
aggressive biological behavior, treatment resistance and poor
survival. Recently, identifying and targeting the GSCs has been
considered as a powerful tool for developing therapeutic
strategies, as GSCs are chemo- and radiotherapy-resistant and
possess self-renewal, differentiation and multi-potentiality, leading
to therapy failure and recurrence.8,9,34 However, this is a difficult
task as majority of the GSC markers described to date, such as
CD133 and nestin, are also present at similar or higher levels in the
adult NSCs. Thus, a cancer therapy targeted using these
ubiquitous and non-specific markers would risk eradicating
reserves of normal self-renewing NSCs. Furthermore, animals
with decreased neurogenesis have impaired performance on
hippocampus-dependent learning tasks,35 suggesting that
ablation of NSCs in the course of glioma therapy might lead to
undesirable cognitive side-effects.
Here, we provided evidence that TRIM11 may act as a novel
marker for malignant gliomas. TRIM11 expression levels correlated
with the glioma malignancy, similar to those of CD133 and
nestin?whose expression levels are indicators for grade levels of
rexessp ttrcoono 2
reh lle 60
rsou rep 40
ty cn 1.5
ilva sbo 1.0
le A 0.5
gliomas and are well-published as prognostic markers.4,5 More
importantly, high TRIM11 were detected only in malignant
gliomaderived GSCs and tissue sections (Table 1, Figure 1a and
Supplementary Figure S1), suggesting that TRIM11 has the
potential to become a specific GSC marker. In addition, TRIM11
levels, concurrent with CD133, were associated with differentiation
status of cells (Figure 2). Concurrence between TRIM11 and CD133
not only occurred at mRNA levels but also at patterns of protein
expression in cells. Co-expression of these two proteins was
detected in both tissue sections (Figure 1b) and GSCs (Figure 1c).
Interestingly, even though CD133 is a cell surface marker,
immunofluorescence staining of CD133 generally shows a diffused
cytoplasmic staining in our study and others,36,37 possibly due to
permeabilization of the cell membranes by fixative that
inadvertently damage surface marker antigens.38
We not only demonstrated for the first time that TRIM11 acts as
a malignant glioma marker but also provided evidence that
TRIM11 may exert its oncogenic function through promoting cell
proliferation (Figures 3a and 4b and Supplementary Figure S3c),
migration (Figure 3b and Supplementary Figure S4a) and invasion
(Figure 3c). Most importantly, our results indicated that TRIM11
exerts its effect through EGFR oncogenic pathway. A decreased
expression of EGFR and weakened activity of Raf/MEK/ERK1/2
MAPK pathway were found in TRIM11 knocked down GBM
cells (Figure 3d and Supplementary Figure S4b), which harbor the
wild-type EGFR gene.39 By contrast, overexpressing TRIM11
increased the expression of EGFR and enhanced the activity of
MAPK pathway (Supplementary Figure S5b).
HB-EGF is a heparin-binding member of the EGF family40 and is
upregulated in response to oncogenesis.41,42 Co-expression of
HBEGF with the amplified EGFR has been found in malignant
gliomas, resulting in the formation of an autocrine loop.43 By
binding and activating EGFR, HB-EGF in turn triggers EGFR
downstream signaling pathways, including MAPKs, which then
induce a rapid and sustained induction of HB-EGF mRNA
expression and secretion of HB-EGF.44 Besides being a ligand for
EGFR, HB-EGF also induces the expression of matrix
metalloproteinases MMP-9 and MMP-3 and elevates activation
of cyclin D1 promoter,45 suggesting a role of HB-EGF as a potent
inducer of tumor growth and angiogenesis. In our study, although
the mRNA levels of EGFR were not affected by TRIM11
downregulation, a significant decrease on HB-EGF and CCND1
mRNA levels was detected in TRIM11 knockdown cells (Table 2
and Figure 3e). By comparison, overexpression of TRIM11
increased the HB-EGF mRNA levels (Supplementary Figures S5c
and d). Further studies will be needed to identify: (
TRIM11 directly regulates the transcription of HB-EGF and/or
CCND1, or alternatively, downregulation of HB-EGF is a
consequence of reduced EGFR levels and weakened MAPK pathway; or
) whether TRIM11 may modulate the process of EGFR
ubiquitination and degradation.
Finally, we provided evidence for the first time that
overexpression of TRIM11 promotes tumorsphere formation in vitro
and tumor growth in vivo (Figure 4). These in vivo results further
support the hypothesis that TRIM11 overexpression predicts the
development of large, vascular and necrotic tumors associated
with poor prognosis.
In summary, our study profiles the gene expression pattern of
TRIM11 in various grade glioma specimens, as well as multiple
GSCs, stable serum-grown glioma cell lines and NSCs, and finds a
strong positive correlation among TRIM11, CD133, and nestin in
high-grade GSCs. In addition, we investigated the oncogenic
function of TRIM11 on proliferation, migration and invasion,
demonstrating that TRIM11 may modulate EGFR expression and
MAPK pathway and bring the first evidence that TRIM11
overexpression promotes glial tumor growth in vivo. This finding
expands our knowledge of the TRIM11 biology from its known
roles in the normal brain development and Alzheimer?s
neurotoxicity into cancer biology, while opening the door for a novel
new area of glioma biology study, with the potential to identify
new translational targets and/or strategies.
MATERIALS AND METHODS
Glioma tissues, cells and media
Approval from the Institutional Review Board was obtained at the
University of California Irvine Medical Center and Children?s Hospital of
Orange County. Surgical specimens of brain tumors (Table 1) were
obtained from patients who had undergone tumor resection with the
neuropathological review completed by a specialty neuropathologist. NSCs
(SC23, SC27, SC30) were derived from the brains of premature neonates by
Dr Philip Schwartz.26 HuTuP01 GBM-GSCs were a gift from Dr David
Panchision (Children?s National Medical Center).46 As previously
described,14 both NSCs and GSCs were cultured in undifferentiated
conditions on Matrigel-coated dishes in 1:1 Dulbecco?s modified Eagle?s
medium (DMEM):F12 medium (Irvine Scientific, Santa Ana, CA, USA),
containing 10% BIT9500 (Stem Cell Technologies, Vancouver, BC, Canada),
292 mg/ml glutamine (Irvine Scientific), 40 ng/ml fibroblast growth factor,
20 ng/ml EGF and 20 ng/ml PDGF. For expansion, one-half of this medium
was replaced every other day, and the cultures were passaged every 7 days
or when confluent using Non-enzymatic Cell Dissociation Solution (Sigma,
St Louis, MO, USA). All our GSCs are able to form spheres when grown on
non-adherent surfaces. The GSCs express glial fibrillary acidic protein when
they are grown under conditions favoring glial differentiation and
bIIItubulin when they are grown under conditions favoring neural
differentiation, confirming their multipotential nature (Supplementary
The stable human glioma cell lines D-54 MG and U-251 MG were gifts
from Dr Darrell Bigner (Duke University, Durham, NC), LN229 was obtained
from Dr Martin Jadus (VA Long Beach Hospital) and U-87 MG as well as
mouse cell line GL261 were gifts from Dr Florence Hofman (University of
Southern California). All the differentiated malignant glioma cell lines were
cultured in a high glucose 1:1 DMEM/F12 medium (Irvine Scientific)
containing 2 mM L-glutamine (Gibco/Invitrogen, Grand Island, NY, USA),
10% fetal bovine serum (Omega Scientific, Tarzana, CA, USA) and 1%
penicillin/streotomycin (Gibco/Invitrogen). All cells were cultured at 37 1C
in a humidified incubator with 5% CO2.
Quantitative reverse-transcriptase?PCR analysis
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD,
USA), and cDNA was generated using the iScript cDNA Synthesis Kit
(Biorad, Hercules, CA, USA). Quantitative PCR reactions (iQ SYBR Green
Supermix, Bio-rad) were conducted using a Bio-Rad CFX96 Real-time
System, and the gene expression levels were normalized to those of ACTB.
The primers were described in Supplementary Methods.
Immunohistochemical and immunofluorescent staining
VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA, USA) was used
for immunohistochemical staining. Formalin-fixed, paraffin-embedded
tissue sections (5 mm) were deparaffinized and rehydrated. After antigen
retrieval, the endogenous peroxidase activity was blocked with 1% H2O2 in
phosphate-buffered saline for 20 min. The sections were incubated with
5% normal goat serum and then exposed to rabbit anti-TRIM11 antibody
(1:40, ProteinTech Group, Chicago, IL, USA) at 4 1C overnight, followed by
incubation with biotinylated anti-rabbit IgG for 1 hour at room
temperature, and counterstained by hematoxylin and eosin. Slides were then
mounted with Permount (Fisher Scientific, Fair Lawn, NJ, USA). For
immunofluorescent double staining, the cells or tissue sections were first
incubated with rabbit anti-TRIM11 and fluorescein
isothiocyanate-conjugated anti-rabbit secondary antibody (Millipore, Billerica, MA, USA) and
then CD133/1 (AC133)-phycoerythrin antibody (Miltenyi Biotec, Auburn,
CA, USA). Nuclei were counterstained with DAPI
(40-6-diamidino-2phenylindole) and slides were mounted with IMMU-MOUNT (Thermo
Scientific, Rockford, IL, USA).
Four human FlexiTube siRNAs targeting TRIM11 (SI00153230, SI00153237,
SI00153244 and SI00153251) and negative control siRNA (1022076) were
purchased from Qiagen and were transfected into cells using HiPerFect
transfection reagent (Qiagen). One siTRIM11 (si-4) was found to knock
down TRIM11 expression most efficiently and was used in most of the
Plasmid construction and transient and/or stable transfection
pcDNA3.1/Zeo Flag-Trim11 and pDsRed2-Trim11 were gifts from Dr Seok
Jong Hong (Northwestern University). Briefly, mouse brain mRNA was used
to amplify full-length TRIM11 by reverse-transcriptase?PCR followed by
cloning into pcDNA3.1/Zeo vector and pDsRed2-C1 vector.24 Transfection
was performed using FuGene 6 (Roche, Indianapolis, IN, USA). For transient
transfection, the cells were analyzed 72 h after transfection. For generation
of stable transfectants, the cells were selected by 0.4 mg/ml of G418
(InvivoGen, San Diego, CA, USA) for 2 weeks.
Antibodies with detailed descriptions are provided in Supplementary
Methods. The images were exposed by KODAK M35A X-OMAT Processor
(EASTMAN KODAK Company, Rochester, NY, USA).
BrdU cell proliferation analysis
Cell proliferation rates were determined by BrdU incorporation according
to the manufacture?s recommendation (Calbiochem, La Jolla, CA, USA).
Briefly, the cells were incubated with BrdU Label for 24 h and then fixed.
Monoclonal anti-BrdU antibody was added, followed by
peroxidaseconjugated secondary antibody. The BrdU incorporation was measured at
dual wavelengths of 450 and 570 nm, using a Model 680 Microplate Reader
Cells (6 103/well) were seeded into 96-well plates. Before testing, MTT
solution (5 mg/ml; 20 ml/well) was added, and cells were incubated at 37 1C
for 5 h. The culture medium was then aspirated and dimethyl sulfoxide
(200 ml/well, Fisher Scientific) was added to dissolve the dark blue crystals.
The absorbance was measured at a wavelength of 570 nm.
Wound closure assay
Cells were plated in 6-well plates and grown to full confluency.
Similarsized wounds were then induced to monolayer cells by scraping a gap
using a micropipette tip. After removing cell debris by rinsing with
phosphate-buffered saline, fresh medium was added, and the cells started
migrating from the edge of the wound and repopulated the gap area. The
time required for ?wound closure? was monitored and photographed
immediately after wound incision and at indicated time points.
Invasion assay was performed using BD BioCoat Matrigel Invasion
Chamber with 8-mm PET membrane (BD Biosciences, Bedford, MA, USA).
Cells were seeded in medium without serum, and medium containing
1.5% fetal bovine serum was used as chemoattractant. After an incubation
of 24 h at 37 1C, non-invasive cells were removed, and invading cells were
fixed with 100% methanol. Cells were then stained in hematoxylin.
Human EGF/PDGF signaling PCR array
Human EGF/PDGF signaling PCR array of 84 genes (SABiosciences, Qiagen,
Frederick, MD, USA) was performed according to the manufacturer?s
instructions. In all, 1 mg of total RNA was used to synthesize cDNA. The
manufacturer?s web-based software package was utilized to calculate fold
changes. Genes with 4twofold differences were considered as significant.
Tumorsphere formation assay
Cells were plated at low density (500/well) in 6-well ultra-low attachment
plates (Corning, Corning, NY, USA). After 2 weeks of culture, the number of
floating tumorspheres in each well (diameter 450 mm) was counted.
Tumorigenicity assay in immunosuppressed mice
Ten 6-week-old BALB/C nu/nu female nude mice were randomly divided
into two groups (n ? 5 in each group): GL261 cells stably transfected with
pDsRed2-C1 vector or pDsRed2-Trim11. Aliquots (100 ml) of 5 105 cells of
each cell group in 25% Matrigel (BD Biosciences) were subcutaneously
injected into nude mice anterior to their right and left thighs on both sides.
After injection, the growth of tumor nodules was estimated with caliper at
a 3-day interval. On day 15 after injection, all mice were killed. Tumors
were removed, and tumor weight was evaluated. The tumor volume was
calculated using the formula V ? (L*W2*p)/6 (L, length; W, width).
Statistical analyses were prepared using GraphPad Prism version 5.04
(GraphPad Software, Inc., La Jolla, CA, USA). All values were presented as
mean?s.e.m. Statistical significance was determined with simple paired
ttests of one-way analysis of variance.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
We thank Xing Gong for the preparation of glioma primary cultures, Dr Ronald C Kim
for the neuropathological diagnosis of tumor samples and Dr Abhishek Chaturbedi
for the organization of the tumor list used in the study. This work was supported by
research funds donated by Ralph and Suzanne Stern and the Community Foundation
of Jewish Federation. This study was also supported, in part, by start-up funds to DAB
from the University of California, Irvine and the UCI Cancer Center Award Number
P30CA062203 from the National Cancer Institute.
Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
1 Gladson CL , Prayson RA , Liu WM . The pathobiology of glioma tumors . Annu Rev Pathol 2010 ; 5 : 33 - 50 .
2 Wen PY , Kesari S. Malignant gliomas in adults . N Engl J Med 2008 ; 359 : 492 - 507 .
3 von Deimling A , Korshunov A , Hartmann C. The next generation of glioma biomarkers: MGMT methylation, BRAF fusions and IDH1 mutations . Brain Pathol 2011 ; 21 : 74 - 87 .
4 Ma YH , Mentlein R , Knerlich F , Kruse ML , Mehdorn HM , Held-Feindt J . Expression of stem cell markers in human astrocytomas of different WHO grades . J Neurooncol 2008 ; 86 : 31 - 45 .
5 Zhang M , Song T , Yang L , Chen R , Wu L , Yang Z et al. Nestin and CD133: valuable stem cell-specific markers for determining clinical outcome of glioma patients . J Exp Clin Cancer Res 2008 ; 27 : 85 .
6 Singh S , Clarke I , Terasaki M , Bonn V , Hawkins C , Squire J et al. Identification of a cancer stem cell in human brain tumors . Cancer Res 2003 ; 63 : 5821 - 5828 .
7 Singh S , Hawkins C , Clarke I , Squire J , Bayani J , Hide T et al. Identification of human brain tumour initiating cells . Nature 2004 ; 432 : 396 - 401 .
8 Chen J , Li Y , Yu TS , McKay RM , Burns DK , Kernie SG et al. A restricted cell population propagates glioblastoma growth after chemotherapy . Nature 2012 ; 488 : 522 - 526 .
9 Bao S , Wu Q , McLendon RE , Hao Y , Shi Q , Hjelmeland AB et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response . Nature 2006 ; 444 : 756 - 760 .
10 Li Z , Wang H , Eyler CE , Hjelmeland AB , Rich JN . Turning cancer stem cells inside out: an exploration of glioma stem cell signaling pathways . J Biol Chem 2009 ; 284 : 16705 - 16709 .
11 Dey M , Ulasov IV , Tyler MA , Sonabend AM , Lesniak MS . Cancer stem cells: the final frontier for glioma virotherapy . Stem Cell Rev 2011 ; 7 : 119 - 129 .
12 Uchida N , Buck DW , He D , Reitsma MJ , Masek M , Phan TV et al. Direct isolation of human central nervous system stem cells . Proc Natl Acad Sci USA 2000 ; 97 : 14720 - 14725 .
13 Zimmerman L , Parr B , Lendahl U , Cunningham M , McKay R , Gavin B et al. Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors . Neuron 1994 ; 12 : 11 - 24 .
14 Gong X , Schwartz PH , Linskey ME , Bota DA . Neural stem/progenitors and glioma stem-like cells have differential sensitivity to chemotherapy . Neurology 2011 ; 76 : 1126 - 1134 .
15 Schmidinger M , Linzmayer L , Becherer A , Fazeny-Doemer B , Fakhrai N , Prayer D et al. Psychometric- and quality-of-life assessment in long-term glioblastoma survivors . J Neurooncol 2003 ; 63 : 55 - 61 .
16 Corn BW , Wang M , Fox S , Michalski J , Purdy J , Simpson J et al. Health related quality of life and cognitive status in patients with glioblastoma multiforme receiving escalating doses of conformal three dimensional radiation on RTOG 98- 03 . J Neurooncol 2009 ; 95 : 247 - 257 .
17 Reymond A , Meroni G , Fantozzi A , Merla G , Cairo S , Luzi L et al. The tripartite motif family identifies cell compartments . EMBO J 2001 ; 20 : 2140 - 2151 .
18 Saurin AJ , Borden KL , Boddy MN , Freemont PS . Does this have a familiar RING? Trends Biochem Sci 1996 ; 21 : 208 - 214 .
19 Borden KL . RING fingers and B-boxes: zinc-binding protein-protein interaction domains . Biochem Cell Biol 1998 ; 76 : 351 - 358 .
20 Hatakeyama S. TRIM proteins and cancer . Nat Rev Cancer 2011 ; 11 : 792 - 804 .
21 Niikura T , Hashimoto Y , Tajima H , Ishizaka M , Yamagishi Y , Kawasumi M et al. A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults . Eur J Neurosci 2003 ; 17 : 1150 - 1158 .
22 Ishikawa H , Tachikawa H , Miura Y , Takahashi N. TRIM11 binds to and destabilizes a key component of the activator-mediated cofactor complex (ARC105) through the ubiquitin-proteasome system . FEBS Lett 2006 ; 580 : 4784 - 4792 .
23 Tuoc TC , Stoykova A . Trim11 modulates the function of neurogenic transcription factor Pax6 through ubiquitin-proteosome system . Genes Dev 2008 ; 22: 1972 - 1986 .
24 Hong SJ , Chae H , Lardaro T , Hong S , Kim KS . Trim11 increases expression of dopamine beta-hydroxylase gene by interacting with Phox2b . Biochem Biophys Res Commun 2008 ; 368 : 650 - 655 .
25 Sanai N , Alvarez-Buylla A , Berger MS . Neural stem cells and the origin of gliomas . N Engl J Med 2005 ; 353 : 811 - 822 .
26 Schwartz PH , Bryant PJ , Fuja TJ , Su H , O'Dowd DK , Klassen H. Isolation and characterization of neural progenitor cells from post-mortem human cortex . J Neurosci Res 2003 ; 74 : 838 - 851 .
27 Kurihara H , Zama A , Tamura M , Takeda J , Sasaki T , Takeuchi T. Glioma /glioblastoma-specific adenoviral gene expression using the nestin gene regulator . Gene Ther 2000 ; 7 : 686 - 693 .
28 Griguer CE , Oliva CR , Gobin E , Marcorelles P , Benos DJ , Lancaster Jr . JR et al. CD133 is a marker of bioenergetic stress in human glioma . PLoS One 2008 ; 3 : e3655 .
29 Zhou XD , Wang XY , Qu FJ , Zhong YH , Lu XD , Zhao P et al. Detection of cancer stem cells from the C6 glioma cell line . J Int Med Res 2009 ; 37 : 503 - 510 .
30 Lim DA , Cha S , Mayo MC , Chen MH , Keles E , VandenBerg S et al. Relationship of glioblastoma multiforme to neural stem cell regions predicts invasive and multifocal tumor phenotype . Neuro Oncol 2007 ; 9 : 424 - 429 .
31 Rao RD , Uhm JH , Krishnan S , James CD . Genetic and signaling pathway alterations in glioblastoma: relevance to novel targeted therapies . Front Biosci 2003 ; 8 : e270 - e280 .
32 Roberts PJ , Der CJ . Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer . Oncogene 2007 ; 26 : 3291 - 3310 .
33 Schlessinger J. Cell signaling by receptor tyrosine kinases . Cell 2000 ; 103 : 211 - 225 .
34 Eramo A , Ricci-Vitiani L , Zeuner A , Pallini R , Lotti F , Sette G et al. Chemotherapy resistance of glioblastoma stem cells . Cell Death Differ 2006 ; 13 : 1238 - 1241 .
35 Zhao C , Deng W , Gage FH . Mechanisms and functional implications of adult neurogenesis . Cell 2008 ; 132 : 645 - 660 .
36 Kordes C , Sawitza I , Muller-Marbach A , Ale-Agha N , Keitel V , Klonowski-Stumpe H et al. CD133 ? hepatic stellate cells are progenitor cells . Biochem Biophys Res Commun 2007 ; 352 : 410 - 417 .
37 Sherman JH , Redpath GT , Redick JA , Purow BW , Laws ER , Jane Jr JA et al. A novel fixative for immunofluorescence staining of CD133-positive glioblastoma stem cells . J Neurosci Methods 2011 ; 198 : 99 - 102 .
38 Fredens K , Dahl R , Venge P. An immunofluorescent method for a specific demonstration of granulocytes and some of their proteins (ECP and CCP) . Histochemistry 1986 ; 84 : 247 - 250 .
39 Bigner SH , Humphrey PA , Wong AJ , Vogelstein B , Mark J , Friedman HS et al. Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts . Cancer Res 1990 ; 50 : 8017 - 8022 .
40 Raab G , Klagsbrun M . Heparin-binding EGF-like growth factor . Biochim Biophys Acta 1997 ; 1333 : F179 - F199 .
41 Suo Z , Risberg B , Karlsson MG , Villman K , Skovlund E , Nesland JM . The expression of EGFR family ligands in breast carcinomas . Int J Surg Pathol 2002 ; 10 : 91 - 99 .
42 Tarbe N , Losch S , Burtscher H , Jarsch M , Weidle UH . Identification of rat pancreatic carcinoma genes associated with lymphogenous metastasis . Anticancer Res 2002 ; 22 : 2015 - 2027 .
43 Mishima K , Higashiyama S , Asai A , Yamaoka K , Nagashima Y , Taniguchi N et al. Heparin-binding epidermal growth factor-like growth factor stimulates mitogenic signaling and is highly expressed in human malignant gliomas . Acta Neuropathol 1998 ; 96 : 322 - 328 .
44 McCarthy SA , Samuels ML , Pritchard CA , Abraham JA , McMahon M . Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes . Genes Dev 1995 ; 9: 1953 - 1964 .
45 Ongusaha PP, Kwak JC , Zwible AJ , Macip S , Higashiyama S , Taniguchi N et al. HBEGF is a potent inducer of tumor growth and angiogenesis . Cancer Res 2004 ; 64 : 5283 - 5290 .
46 Pistollato F , Chen H-L , Rood BR , Zhang H-Z , D 'Avella D , Denaro L et al. Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma . Stem Cells 2009 ; 27 : 7 - 17 .