Download MIF Maintains the Tumorigenic Capacity of Brain

Published OnlineFirst March 15, 2016; DOI: 10.1158/0008-5472.CAN-15-1011
Cancer
Research
Tumor and Stem Cell Biology
MIF Maintains the Tumorigenic Capacity of Brain
Tumor–Initiating Cells by Directly Inhibiting p53
Raita Fukaya1, Shigeki Ohta2, Tomonori Yaguchi3, Yumi Matsuzaki2, Eiji Sugihara4,
Hideyuki Okano2, Hideyuki Saya4,Yutaka Kawakami3, Takeshi Kawase1, Kazunari Yoshida1,
and Masahiro Toda1
Abstract
Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we
isolated primary cells from 21 human brain tumor specimens to
establish cell lines with high tumorigenic potential and to identify
the molecules enabling this capability. The morphology, sphereforming ability upon expansion, and differentiation potential of
all cell lines were indistinguishable in vitro. However, testing for
tumorigenicity revealed two distinct cell types, brain tumor–
initiating cells (BTIC) and non-BTIC. We found that macrophage
migration inhibitory factor (MIF) was highly expressed in BTIC
compared with non-BTIC. MIF bound directly to both wild-type
and mutant p53 but regulated p53-dependent cell growth by
different mechanisms, depending on glioma cell line and p53
status. MIF physically interacted with wild-type p53 in the nucleus
and inhibited its transcription-dependent functions. In contrast,
MIF bound to mutant p53 in the cytoplasm and abrogated
transcription-independent induction of apoptosis. Furthermore,
MIF knockdown inhibited BTIC-induced tumor formation in a
mouse xenograft model, leading to increased overall survival.
Collectively, our findings suggest that MIF regulates BTIC
function through direct, intracellular inhibition of p53, shedding
light on the molecular mechanisms underlying the tumorigenicity
of certain malignant brain cells. Cancer Res; 76(9); 1–11. 2016 AACR.
Introduction
contribute significantly to the tumor mass, but not to the initiation and maintenance of the tumor. BTICs are thus considered a
promising target for future therapies.
Macrophage migration inhibitory factor (MIF) was originally
identified as a proinflammatory cytokine, but was later found to
be a critical regulator of tumor progression as well (14–18). MIF
upregulation has been observed in human melanoma (19), lung
cancer (20), prostate cancer (21), hepatocellular carcinoma (22),
and glioblastoma (23–27). MIF expression was significantly
higher in the tumor tissues of glioblastoma patients than in
normal brain tissues (24, 25). The precise roles of MIF in the
initiation and progression of glioma, however, remain unclear.
MIF plays numerous roles through receptor-mediated signaling
pathways that are activated by cell surface receptors, such as CD74,
CD44, and CXCRs (28–30), or by intracellular interactions
through binding to JAB1 (31). MIF has also been shown to
function as a p53 inhibitor (16) that directly binds and antagonizes p53 function in the cells (32).
The p53 protein is an important and well-characterized tumor
suppressor (33). In response to various forms of cell stress, p53
regulates diverse target molecules, which may induce cell-cycle
arrest and apoptosis in a transcription-dependent manner (34).
p53 has also been shown to possess other biologic activities,
including induction of apoptosis, in mitochondria in a transcription-independent manner (35–37).
In this study, we show that MIF expression is significantly
higher in BTICs isolated from human glioma tissues than in
non-BTICs from the same tissue source and that MIF acts as a
direct intracellular p53 inactivator that regulates cell proliferation
and apoptosis in glioma cells. In addition, we highlight the link
between the roles of MIF and tumor-initiating cells associated
with p53, a candidate for the dedifferentiation barrier.
Glioblastoma, the most common form of primary malignant
brain tumor, is nearly always fatal (1). Although neurosurgeons
may be able to remove the main tumor mass, this procedure
typically leaves behind a small population of tumor cells that
retain the capacity for tumorigenesis (2). For this reason, glioblastomas almost always relapse, highlighting the need for more
precisely targeted therapeutic approaches. A number of recent
studies have demonstrated that tumors are composed of cells with
functional heterogeneity that form a hierarchy, especially in the
context of tumor initiation (3, 4). Characterized by its heterogeneous tumor types (5–8), glioblastoma is now also thought to be
initiated and maintained by a subpopulation of tumor cells,
called brain tumor stem cells or brain tumor–initiating cells
(BTIC; refs. 9–13). The clinical significance of BTICs is emphasized by the strongly enhanced capacity of these cells to initiate
brain tumors, in contrast to the majority of tumor cells, which may
1
Department of Neurosurgery, Keio University School of Medicine,
Tokyo, Japan. 2Department of Physiology, Keio University School of
Medicine, Tokyo, Japan. 3Division of Cellular Signaling, Institute for
Advanced Medical Research, Keio University School of Medicine,
Tokyo, Japan. 4Division of Gene Regulation, Institute for Advanced
Medical Research, Keio University School of Medicine, Tokyo, Japan.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Masahiro Toda, Keio University School of Medicine, 35
Shinanomachi, Shinjuku, Tokyo 1608582, Japan. Phone: 813-5363-3807; Fax:
813-3358-0479; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-15-1011
2016 American Association for Cancer Research.
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Fukaya et al.
Table 1. Isolation of BTICs
hG002
hG016
hG001
hG004
hG006
hG008
hG010
hG011
hG013
hG014
hG015
hG017
hG018
hG020
hG021
hG007
hG005
hG012
hG019
hG003
hG009
Tumor type
Pilocytic astrocytoma
Anaplastic astrocytoma
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma (Gliosarcoma)
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma
Glioblastoma
Oligodendroglioma
Anaplastic oligodendroglioma
Anaplastic oligodendroglioma
Anaplastic oligodendroglioma
Medulloblastoma
Olfactory neuroblastoma
Age (y)
29
43
46
64
25
61
50
40
39
37
81
37
85
53
73
30
81
36
41
34
54
Sex
M
M
M
F
F
F
F
M
F
F
F
F
F
M
M
M
F
F
M
M
M
Culture
Impossible
Temporarily
Temporarily
Temporarily
Impossible
Permanently
Temporarily
Temporarily
Temporarily
Temporarily
Temporarily
Temporarily
Temporarily
Permanently
Impossible
Temporarily
Temporarily
Temporarily
Permanently
Temporarily
Impossible
Differentiation
potential
Inoculation of
the tumor cells
In vivo tumor
formation
þ
Not tested
þ
Tested
Not tested
Not tested
þ
þ
þ
þ
þ
þ
þ
þ
þ
Tested
Tested
Tested
Tested
Tested
Tested
Tested
Tested
Tested
þ
þ
þ
þ
þ
þ
þ
Tested
Not tested
Tested
Tested
Tested
þ
P53
M
WT
WT
Abbreviations: M, mutant; WT, wild-type; þ, possess the potential; , none of the potential.
Materials and Methods
Human tissues and cell culture
Approval to use human neurosphere cultures was obtained
from the Keio University ethics committee. Tissue procurement
procedures were performed in accordance with the Declaration of
Helsinki and in compliance with the ethical guidelines of the
Japan Society of Obstetrics and Gynecology and the Network of
European CNS Transplantation and Restoration. Brain tumor
samples were obtained from patients undergoing surgery at Keio
University Hospital (Tokyo, Japan) following approval from the
Institutional Review Board at Keio University School of Medicine
(Tokyo, Japan). Tumor samples were mechanically and enzymatically dissociated into single cells. All tumor-derived cells and
neural stem cells (NSC) were cultured at 37 C in a humidified
incubator with 5% CO2 in neurosphere medium (NSM) consisting of Neurobasal Medium (Invitrogen), B27 (Invitrogen),
human recombinant bFGF and EGF (PeproTech, 20 ng/mL each),
and heparin (10 ng/mL; Sigma). U87MG and T98G cells were
provided by Dr. Y. Ohashi (Keio University) and maintained in
DMEM (Wako) supplemented with 10% FBS. Short tandem
repeat DNA profiling of T98G and U87MG cells was performed
using the Cell ID System (Promega) in August 2011. Human
astrocytes were obtained from Lonza and maintained in Astrocyte
Basal Medium (Lonza).
Animal models
For the brain tumor initiation assay, 6- to 8-week-old female
NOD/SCID mice were purchased from Sankyo Lab. Samples of
1,000, 10,000, or 100,000 primary cells cultured from brain
tumor tissues were stereotactically injected into the brain striatum
(2.5 mm right of the midline, 3 mm deep). The mice were killed
when they exhibited neurologic symptoms or at 150 days after
inoculation, whichever came first. All procedures involving animal experiments were approved by the Animal Care and Use
Committee of the Keio University School of Medicine (Tokyo,
Japan). To evaluate the tumor-initiating potential of BTICs by
treatment of lentivirus expressing MIF shRNA, two groups of mice
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were prepared. hG008 cells were infected with lentivirus expressing either MIF shRNA or control shRNA [multiplicity of infection
(MOI) ¼ 1] for 24 hours and washed twice in PBS, after which the
number of live cells was counted. A total of 10,000 cells were
inoculated into the brain striatum (n ¼ 5); the mice were killed
and tumor generation was evaluated by histologic examinations
six weeks after implantation. For the treatment study using
lentivirus, intracranial transplantation of BTICs was performed
as described above. To establish glioma xenografts for lentivirusmediated MIF shRNA treatment, hG008 cells were injected into
the brains of NOD/SCID mice (3,000 cells/mouse, eight mice/
group). Lentivirus expressing either MIF shRNA or shRNA control
(MOI ¼ 1) was injected twice into the tumor sites at two and three
weeks after tumor cell implantation. The effects of MIF shRNA
treatment were evaluated to compare the survival times of each
group by Kaplan–Meier analysis.
Immunoprecipitation assay
Immunoprecipitation was performed using an nProtein ASepharose 4 Fast Flow Kit (GE Healthcare) following the manufacturer's instructions. Nuclear and cytoplasmic extracts of tumor
cells (1 mg) were suspended in 1 mL RIPA buffer (Pierce) and
coupled to 50 mL nProtein A-Sepharose (GE Healthcare) for 1
hour at 4 C for precleaning. The supernatant was incubated with
mouse anti-p53 (DO-1; 1:200; Santa Cruz Biotechnology) or
nonspecific mouse IgG (sc-2025; Santa Cruz Biotechnology)
antibody for 4 hours at 4 C. A total of 50 mL nProtein A-Sepharose
was added to the samples, which were incubated for 1 hour at 4 C.
The immune complexes were washed twice with RIPA buffer and
once with wash buffer (50 mmol/L Tris, pH 8.0). The beads were
resuspended in the sample buffer (1% SDS, 100 mmol/L DTT, 50
mmol/L Tris, pH 7.5) and boiled for 5 minutes before the supernatants were subjected to Western blot analysis.
Gel shift assay
Gel shift assays were performed using a P53 EMSA Kit
(Panomics) following the manufacturer's instructions. In each
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MIF Maintains the Tumorigenic Capacity of BTICs
Figure 1.
Characteristics of BTICs. A, tumor
spheres expressing nestin. Scale bar,
100 mm (left); 50 mm (right). B, BTIC
spheres can differentiate into three cell
lineages: glial, neural, and
oligodendroglial cells. Scale bar, 50 mm.
C, typical hematoxylin and eosin
staining images of BTICs implanted in
NOD/SCID mice. In mouse brain, BTICgenerated tumors were highly
infiltrative and hemorrhagic, features
characteristic of human glioblastoma.
Scale bar, 2 mm (left); 100 mm (right).
sample, 10 mg of protein from nuclear extracts isolated from
U87MG cells was incubated with biotinylated p53 consensus
binding sequence oligonucleotides (AY1032P; Panomics). Separation of bound and free probe was achieved by electrophoresis using a 6.0% nondenaturing polyacrylamide gel, and the
protein–DNA complexes were transferred to a nylon membrane
(Hybond-N, GE Healthcare). The DNA was crosslinked to the
membrane using a UV cross-linker. Protein–DNA complexes
were detected using streptavidin–horseradish peroxidase and
visualized after exposure to Hyperfilm ECL film (Amersham
Biosciences). Experiments were performed independently at
least twice.
Statistical analysis
Statistical analyses were performed using Statcel2. Student t
tests were performed to determine the statistical significance of the
quantitative PCR study, cell proliferation study, double knockdown study, reporter gene assay, and apoptosis assay. Mann–
Whitney tests were used to assess in vivo tumor initiation after
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treatment with shRNAs. P < 0.05 was considered to represent
significance.
All other methods are available in Supplementary Material and
Methods.
Results
Isolation and characterization of BTICs
Twenty-one brain tumors were dissociated into single cells and
plated at a clonal density (38) in NSM. These brain tumors and
their characteristics are shown in Table 1. Four of the tumors,
including two glioblastomas, one pilocytic astrocytoma, and one
olfactory neuroblastoma, could not be cultured in NSM. Most of
the primary cells could be cultured temporarily as glioma spheres
but did not culture for more than 8 passages. Only three cultures,
hG008, hG019, and hG020, could be cultured for more than 20
passages and proliferate permanently. These three cell lines
included two glioblastomas, hG008 and hG020, and one anaplastic oligodendroglioma, hG019. Notably, hG008 and hG019
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Figure 2.
MIF expression in glioma cells and tissues. A,
MIF gene expression was significantly higher
in BTICs (hG008, hG019, and hG020)
compared with non-BTICs (hG012, hG013,
and hG014; mean SD). , P < 0.01. B,
immunohistochemical analysis of MIF
expression in human glioblastoma tissues.
MIF was expressed heterogeneously in tumor
cells (top; N, necrotic region). Strong MIF
expression was observed in both the nucleus
and cytoplasm, and most of the MIFexpressing tumor cells showed nuclear
hypertrophy in glioblastoma tissues
(bottom). Scale bar, 200 mm. C, protein
expression of MIF and p53 in the cytoplasmic
and nucleic fractions.
were obtained from tumors that had recurred after surgery,
radiation, and chemotherapy. hG020 was isolated from a patient
who had undergone surgery for a newly diagnosed glioblastoma.
Multiple point mutations in the DNA-binding domain of the p53
gene were found in hG008 (Supplementary Table S1). Wild-type
p53 was expressed in hG019 and hG020. BTICs usually show
potential for tumorigenesis, proliferation, self-renewal, and multidifferentiation (39). These three expandable primary cells generated glioma spheres and expressed nestin, a marker for NSCs
(Fig. 1A). To assess their multilineage differentiation potential,
the primary cells were cultured in differentiation medium containing 1% FBS without EGF and FGF2. All of the analyzed
primary cells, including BTICs and non-BTIC, were able to differentiate into cells of three neural lineages: glial (GFAP), neuronal (b-III tubulin), and oligodendroglial (O4; Fig. 1B; Table 1).
Next, to determine whether the primary cells were tumorigenic in
vivo, the primary cells were xenografted into the striatum of NOD/
SCID mice. We attempted to generate tumors in the mouse brain
by inoculating 1,000, 10,000, or 100,000 primary cells and
observed tumorigenesis solely from hG008, hG019, and hG020
cells (Fig. 1C; Table 1), from which we concluded that hG008,
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hG019, and hG020 were BTICs. The other primary cells, which did
not generate brain tumors in vivo but showed multilineage differentiation potential and limited potential for expansion in vitro,
were defined as non-BTICs.
BTICs expressed MIF and p53
To identify molecules involved in brain tumorigenesis, we
analyzed gene expression in BTICs and non-BTICs. MIF is a
cancer-related gene that has previously been shown to promote
cell survival and proliferation in mouse NSCs (40). Intriguingly,
the MIF (Fig. 2A) gene was more highly expressed in BTICs than in
non-BTICs, astrocytes, and NSCs. Next, MIF expression in human
glioblastoma tissues was assessed by immunohistochemical analysis. Some tumor cells expressed MIF in both the cytoplasm and
the nucleus (Fig. 2B). MIF was highly expressed in BTICs and
glioblastoma tissues, although the gene expression level of CD74
and CXCR4, known as MIF direct binding receptors, was lower in
cultured BTICs than in NSCs (Supplementary Fig. S1). We decided
to focus on the intracellular functions of MIF in this study. MIF
expression was further investigated in the cytoplasm and nucleus
by immunoblotting (Fig. 2C). MIF protein expression was verified
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MIF Maintains the Tumorigenic Capacity of BTICs
in the cytoplasmic and nucleic fractions in BTICs and glioblastoma cell lines (T98G, U87MG). Intriguingly, MIF expression was
low in the cytoplasm and was not detected in the nuclei of NSCs
and astrocytes. MIF has previously been identified as a molecule
that inhibits p53 function (16), so we investigated p53 expression. The cytoplasmic fractions of hG008, hG019, and T98G
displayed p53 expression, whereas hG020, NSCs, and U87MG
showed very low levels of p53 expression. In the nuclear fractions,
p53 expression was high in BTICs and T98G and moderate in
NSCs, astrocytes, and U87MG (Fig. 2C).
MIF binds to p53 and inhibits p53 function
We first examined the role of MIF in two glioblastoma cell lines,
U87MG and T98G. Both cell lines expressed MIF and p53, but
these exhibit different p53 gene mutation statuses: U87MG
express wild-type p53, and T98G has a homozygous mutation
at codon 237 (M ! I) of p53 (41). MIF gene expression
was knocked down using MIF siRNA in U87MG, and cell proliferation was assessed. U87MG cells treated with MIF siRNA
showed decreased cell proliferation compared with siRNA controls (Fig. 3A). Next, we tested the role of p53 in this system. A p53
knockdown system was established using p53 siRNA in U87MG
and T98G cells (Supplementary Fig. S2), and we found that p53
gene silencing abrogated the decrease in cell proliferation induced
by MIF gene silencing in U87MG cells (Fig. 3B and Supplementary
Fig. S2). This suggests a strong functional relationship between
MIF and p53. We next explored the physical interaction between
MIF and p53 in U87MG cells. Immunoprecipitation analysis was
performed using U87MG nuclear protein, and we confirmed that
p53 binds directly to MIF in the nuclei of U87MG (Fig. 3C). To
investigate the regulation of p53 target genes under conditions in
which MIF expression is suppressed, reporter gene assays were
performed using p21-Luc and Bax-Luc reporter plasmids. The
luciferase activities of P21 and BAX were elevated by MIF siRNA
treatment compared with controls in U87MG (Fig. 3D). Immunoblot studies also confirmed upregulation of BAX and P21
protein in a dose-dependent manner by MIF siRNA treatment in
U87MG cells (Fig. 3E). Cell-cycle analysis was performed by flow
cytometry and G1 cell-cycle arrest was observed in U87MG in
response to MIF siRNA treatment (Fig. 3F). In addition, a caspase3/7 activity assay showed a dose-dependent increase in apoptosis
following MIF siRNA treatment (Fig. 3G). Taken together, these
results indicate that MIF downregulation induces cell-cycle arrest
and apoptosis in U87MG cells.
To elucidate the functional relationship between p53 and MIF,
changes in MIF-regulated p53 DNA-binding ability was assessed
by gel shift assay. A p21 promoter DNA probe containing a p53binding region was used in a gel shift assay using nuclear protein
isolated from U87MG cells transfected with either MIF siRNA or
control siRNA. The gel shift assay showed that p53-specific binding was upregulated by MIF siRNA compared with the control
U87MG cells (Fig. 3H). MIF has thus been shown to bind to p53 in
the nucleus of U87MG cells, and MIF siRNA-mediated gene
silencing increased p53 DNA-binding ability, cell-cycle arrest,
and apoptosis. We also assessed MIF function in T98G cells in
the same manner as performed in U87MG cells and observed that
MIF gene silencing by siRNA treatment decreased cell proliferation compared with the controls in a dose-dependent fashion
(Fig. 4A and B). This reduction in cell proliferation was also p53
dependent in T98 cells, as seen in U87MG cells (Fig. 4C and
Supplementary Fig. S2). However, in contrast to the results from
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U87MG cells, we did not observe an increase in the transcriptional
activity of the p21 and BAX promoters following MIF siRNA
treatment in the reporter gene assay, suggesting that mutated
p53 does not activate the transcription of target genes in T98G
cells (Supplementary Fig. S3A).
We therefore focused on the function of p53 in the cytoplasmic
fraction of T98G cells and observed that p53 binds directly to MIF
in this fraction (Fig. 4D). MIF siRNA treatment led to the induction of caspase-3/7 activity (Fig. 4E) without the induction of cellcycle arrest in T98G cells (Supplementary Fig. S3B). We hypothesized that the decrease in cell proliferation induced by MIF gene
silencing in T98G is regulated by proapoptotic events induced by
p53 in the cytoplasmic fraction. In fact, MIF gene silencing led to
the accumulation of p53 in the mitochondria fraction (Fig. 4F),
which may lead to increased apoptosis in mitochondria. Moreover, treatment with PFT-m, a known inhibitor of p53 translocation to mitochondria, abrogated the suppression in proliferation
induced by MIF gene silencing in T98G cells (data not shown).
These findings underscore the importance of MIF-dependent/
induced p53 translocation to the mitochondria, which induces
apoptosis in T98G cells. Collectively, these results show that MIF
is able to bind to both wild-type and mutant p53, and to support
cellular proliferation in a p53-dependent manner in both U87MG
and T98G cells, although the underlying mechanisms differ
between the two glioma cell lines.
MIF is the therapeutic target for BTICs
To confirm the effects of MIF on proliferation and apoptosis in
BTICs, a lentivirus expressing MIF shRNA (lenti-shMIF) was
employed, and the efficacy of the lentivirus was assessed by
immunoblot analysis in hG008 cells, which confirmed that
changes had occurred in p53 protein expression (Fig. 5A). We
evaluated the effects of lenti-shMIF on cell proliferation in three
BTIC lines (hG008, hG019, and hG020). Dose-dependent suppression of cell proliferation was observed in all BTICs on lentishMIF treatment (Fig. 5B). Microscopic observations revealed that
lenti-shMIF infection decreased cell survival and inhibited glioma
sphere formation compared with the control (Fig. 5C). Suppression of cell proliferation by MIF was also supported by p53 in
hG008 cells (Supplementary Fig. S4).
We further analyzed the gene expression change of p21 and
BAX, known as transcription-dependent downstream targets of
p53, in two BTICs (hG008, p53 mutant-type; hG020, p53 wildtype), U87MG (p53 mutant-type), and T98G (p53 mutant-type)
to investigate the effect of MIF knockdown. p21 and BAX genes
were upregulated on infection with lenti-shMIF in hG020 and
U87MG compared with hG008 and T98G in quantitative PCR
study (Supplementary Fig. S5A and S5B). We also confirmed that
MIF gene silencing led to the accumulation of p53 in the mitochondria fraction in hG008, similar to the effect in T98G (Supplementary Fig. S5C). These results suggested that MIF inhibits the
function of p53 in transcription-dependent and -independent
manner, based on the status of the p53 gene in glioma cells and
BTICs, respectively. Lenti-shMIF treatment of hG008 and hG020
cells also led to an increase in caspase-3/7 activity in a dosedependent manner (Supplementary Fig. S5D).
We next examined the therapeutic effect of MIF targeting using
mouse xenograft models. First, 1 104 hG008 cells infected with
either lenti-shMIF or lenti-shControl were implanted into the
brains of immunodeficient NOD/SCID mice, and tumorigenicity
was evaluated in each group at 42 days after implantation. The
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Figure 3.
MIF binds to p53 in the nucleus and inhibits p53 function in a transcription-dependent manner in U87MG cells. A, proliferation of U87MG cells was suppressed
by MIF siRNA (siMIF) treatment compared with the controls (sicont.; mean SD). , P < 0.01; , P < 0.001. B, the ability of MIF siRNA knockdown to suppress
cell proliferation was abrogated by p53 gene silencing in U87MG cells at 7 days after transfection (mean SD). , P < 0.05. C, MIF binds to p53 in
the nucleic fraction of U87MG cells. (Continued on the following page.)
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MIF Maintains the Tumorigenic Capacity of BTICs
Figure 4.
MIF binds to p53 in the cytoplasmic fraction of T98G cells and inhibits the apoptotic function of p53. A, decreases in MIF protein expression by gene silencing
did not affect p53 protein expression at 3 days after MIF siRNA (siMIF) transfection. B, cell proliferation was suppressed by MIF siRNA transfection in a dosedependent manner (mean SD). , P < 0.05; , P < 0.001. C, the ability of MIF siRNA to suppress cell proliferation was abrogated by p53 gene silencing
at 7 days after transfection (mean SD). , P < 0.001. D, MIF physically bound to p53 in the cytoplasmic fraction of T98G cells. E, MIF gene silencing in T98G cells led
to an increase in caspase-3/7 activity compared with that of U87MG cells, 3 days after MIF siRNA transfection (mean SD). , P < 0.05; , P < 0.001. F,
quantitation of p53 protein in mitochondria fraction by Western blot (WB) analysis showed increased p53 protein localization in the mitochondria of T98G cells
following MIF gene silencing by infection with a lentivirus expressing MIF shRNA at 3 days after infection (mean SD). , P < 0.05. IP, immunoprecipitate.
implantation of hG008 cells infected with the control virus led to
brain tumor formation in 4 of 5 mice, whereas no tumors were
observed in mice implanted with lenti-shMIF–infected hG008
cells (n ¼ 5), suggesting that MIF may be an essential factor in
maintenance for brain tumors in this experimental system
(Fig. 5D). Next, we attempted to validate the therapeutic effect
of MIF gene silencing in BTICs. First, 1 103 hG008 cells were
implanted in the brains of NOD/SCID mice and then a lentiviral
vector expressing either MIF-shRNA or control-shRNA was administered to the BTIC implantation site on days 7 and 14
(Continued.) D, the transcriptional activities of cell-cycle regulator p21 and apoptotic factor BAX were elevated by MIF gene silencing at 3 days after transfection
(mean SD). , P < 0.05; , P < 0.001. E, BAX and p21 protein expression was induced by MIF gene silencing in U87MG cells at 3 days after siRNA transfection.
F, G1 arrest was induced by MIF gene silencing at 3 days after siRNA transfection. G, caspase-3/7 activity was increased at 3 days after MIF siRNA
transfection in a dose-dependent manner (mean SD). , P < 0.05; , P < 0.001. H, the physical binding between a p53-specific DNA probe and nuclear
extracts from U87MG cells was increased by MIF gene silencing. These physical interactions were antagonized by a cold probe and an anti-p53 antibody.
The nuclear extracts were prepared from U87MG cells transfected with different siRNA concentrations at 3 days after transfection. IP, immunoprecipitate; LA,
luciferase; WB, Western blotting.
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Figure 5.
MIF regulates the initiation of tumorigenesis in BTICs, and MIF gene silencing shows a therapeutic effect on BTICs in a xenograft model. A, hG008 cells infected with
lenti-shMIF showed a significant decrease in MIF protein expression compared with controls (lenti-shControl) without affecting p53 protein expression at
3 days after infection. B, BTICs infected with lenti-shMIF inhibited cell proliferation in a virus dose-dependent manner at 7 days after infection (mean SD). , P < 0.05;
, P < 0.01; , P < 0.001. C, hG008 cells infected with lenti-shMIF did not form tumor spheres, whereas control cells grew while forming healthy tumor
spheres. Scale bar, 200 mm. D, hG008 cells infected with lenti-shMIF did not generate brain tumors in mice, whereas 4 of 5 mice injected with hG008 cells infected
with control virus generated brain tumors at 42 days after cell implantation (mean SD; P ¼ 0.01). E, the brain tumor–bearing mice implanted with hG008
cells were infected with either lenti-shMIF (n ¼ 8) or shControl (n ¼ 8) virus at two and three weeks after cell implantation. Mice treated with lenti-shMIF showed
longer survival compared with control mice, as evaluated by Kaplan–Meier analysis (mean SD; P ¼ 0.04).
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MIF Maintains the Tumorigenic Capacity of BTICs
postimplantation. Survival periods were evaluated for each therapeutic group, and the mice inoculated with lenti-shMIF lived
significantly longer than the control mice (n ¼ 8/group, P ¼
0.04; Fig. 5E and Supplementary Fig. S6). These results suggest
that MIF plays a key role in the proliferation and tumorigenesis of
BTICs, both in vitro and in vivo.
Discussion
High-viability brain tumor cells are known to be present in
the restricted ring–enhanced region surrounding the tumor
mass on gadolinium-enhanced MRI (42). Indeed, the efficiency of isolation of BTICs from glioma tissues is known to be
quite low. One study showed that nine cell lines established
from 19 brain tumor tissues could be cultured for more than
eight passages as tumor spheres and that only six of the nine
cell lines were transplantable into mouse brains for examination of their tumorigenic potential (43). In this study, we
identified only three BTICs from 21 brain tumor masses, which
we have compared in the current study to non-BTICs that
showed no capacity for tumorigenicity. Previous BTIC studies
have distinguished primary tumor cell types based on their
expression of molecular markers, such as CD133. There are,
however, no previous studies that distinguish BTICs and nonBTICs based on their functional properties. Therefore, the
identification of high MIF expression in BTICs is important
to gain a better understanding of the mechanisms underlying
tumorigenesis.
We first investigated the functions of MIF in two glioblastoma
cell lines, U87MG and T98G, as these were available for most
reproducible examinations, have well-known biologic properties,
including the status of the p53 gene, and also exhibit tumorinitiating potential.
We found high levels of MIF expression in the nuclei and
cytoplasm of human glioma cells, but low expression of CD74,
a direct binding receptor of MIF (29). A previous study reported
scant expression of CD74 in several glioma cells (44). On the
basis of the intracellular distribution of MIF in glioma cells, we
focused on physical interactions between MIF and p53. Similar
to the distinction observed the mutation status of p53, the
intracellular distribution of p53 differed between U87MG
and T98G cells. p53 was expressed in both the nucleus and
cytoplasm of T98G cells, but only in the nucleus of U87MG
cells. Our findings suggest that MIF exerts its functions
through association with p53 mainly in the nucleic fraction
of U87MG cells in a transcription-dependent manner. In T98G,
MIF may inhibit transcription-independent apoptosis by binding to cytoplasmic p53, as the mutant p53 lacks transcriptionactivating ability (Supplementary Fig. S7). It has recently been
reported that p53 exerts various biologic functions, including
involvement in mitochondrial apoptosis in a transcriptionindependent fashion (35–37). In most cells with p53 mutations, including T98G and hG008 cells, point mutations are
located in the DNA-binding domain of p53, leading to a
complete lack or decrease in DNA-binding ability. In the
cytoplasm of some cell types, mutant p53 has recently been
shown to induce apoptosis through a transcription- independent pathway more effectively than wild-type p53 (45, 46),
which is consistent with our results (U87MG vs. T98G cells,
hG020 vs. hG008 cells). The decrease of cell proliferation
following MIF gene silencing was abrogated by p53 gene
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knockdown both in glioma cells and BTICs, indicating that
the MIF–p53 axis was essential for glioma tumorigenesis based
on BTICs. A more detailed analysis of MIF function in BTICs
may thus be important, especially when considering the development of drugs targeting MIF.
Histologically immature neoplasms are generally more
malignant and aggressive in their development (47). Indeed,
BTICs are assumed to be the origin of brain tumors and also to
remain in an extremely undifferentiated status. Recently, it has
been reported that p53 acts as a barrier to somatic cell reprogramming, a dedifferentiation process resembling tumor formation (48). Our observations suggest the following hypothesis: if p53 in a normal glial cell is directly inactivated by
accumulated MIF by factors such as cell stresses, including
hypoxia or hypoglycemia, the cell may lose its resistance to
dedifferentiation and develop into an immature tumor cell
resembling a BTIC, the cell of origin and driving force of tumor
growth. In this hypothesis, BTICs may be derived from fully
differentiated cells, such as astrocytes, not only from NSCs in
the brain. This possibility is supported by at least one recent
study (49). Additional evidence also suggests that glioblastoma
develops largely in subcortical white matter and rarely in
ventricular zone, in which NSCs are abundantly present in
adult human brain (50).
In conclusion, we have shown that MIF is highly expressed in
BTICs and plays critical roles in the tumorigenicity of BTICs in vivo.
Our study further suggests that MIF plays an important role in
transforming benign glial cells to BTICs in the niche region, where
glial cells are more likely to be exposed to cellular stresses.
Moreover, MIF may represent a molecular therapeutic target and
clinical marker for glioma therapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interests were disclosed.
Authors' Contributions
Conception and design: R. Fukaya, S. Ohta, K. Yoshida, M. Toda
Development of methodology: R. Fukaya, S. Ohta
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): R. Fukaya, S. Ohta, T. Yaguchi, Y. Matsuzaki
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): R. Fukaya, S. Ohta, T. Yaguchi, E. Sugihara
Writing, review, and/or revision of the manuscript: R. Fukaya, S. Ohta,
H. Okano, H. Saya, Y. Kawakami, K. Yoshida, M. Toda
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): R. Fukaya, S. Ohta, Y. Matsuzaki, H. Okano
Study supervision: S. Ohta, Y. Kawakami, T. Kawase, K. Yoshida, M. Toda
Acknowledgments
The authors thank Dr. C. Ishioka (Tohoku University) for providing the p21Luc and Bax-Luc pMX-Ig vectors, Dr. Y. Kanemura for providing human NSCs,
and Prof. Douglass Sipp (Keio University) for invaluable comments for the
manuscript.
Grant Support
This work was supported by JSPS KAKENHI grant no. 22791356 (R. Fukaya)
and MEXT Global COE program project base no. F14 (H. Okano).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received April 15, 2015; revised February 19, 2016; accepted March 4, 2016;
published OnlineFirst March 15, 2016.
Cancer Res; 76(9) May 1, 2016
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Fukaya et al.
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MIF Maintains the Tumorigenic Capacity of Brain Tumor−
Initiating Cells by Directly Inhibiting p53
Raita Fukaya, Shigeki Ohta, Tomonori Yaguchi, et al.
Cancer Res Published OnlineFirst March 15, 2016.
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