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Abstract

Epithelial‑mesenchymal transition (EMT) is a reversible biological process that occurs in epithelial cells. EMT ultimately leads to the acquisition of a mesenchymal phenotype, characterized by increased cell motility and resistance to genotoxic agents. These processes mostly overlap with the acquirement of stem cell properties in differentiated tumor cells. With regard to gliomas, the clinical picture is heterogeneous, even within the same grades and histological categories of the disease. Furthermore, the areas of invasion and responses to radiochemotherapy are markedly different among cases, and occasionally even in the same patient. Such phenotypic diversity in glioma tissues may be caused by various microenvironmental factors, as well as intrinsic genetic alterations. The current review focuses on the EMT‑inducing factors that are present in gliomas; these typically vary from those observed in epithelial cancers, as no basement membrane is present. Furthermore, the most important cell‑cell contact factor, E‑cadherin, is rarely expressed in gliomas. The microenvironment that induces EMT in gliomas is characterized by hypoxia and the enrichment of myeloid cells following stimulation by transforming growth factor‑β. Anti‑vascular endothelial growth factor therapy, including the use of bevacizumab, may be a suitable candidate to modulate the glioma microenvironment.

Introduction

Epithelial-mesenchymal transition (EMT) is a
biological process in which polarized epithelial cells are induced
to undergo numerous biochemical changes; this results in a
mesenchymal phenotype, defined by an enhanced migratory capacity
and elevated resistance to genotoxic agents (1,2). EMT is
indispensable for wound healing, embryonic development and tissue
remodeling. As a pathological process, EMT also induces migratory
and invasive capabilities in epithelial tumor cells without a loss
in viability (1,2). The process of EMT includes the
detachment of tumor cells from the basement membrane. Although the
central nervous system (CNS) lacks this critical tissue component,
key invasive mechanisms overlap between cancers of the CNS and
other cancer types (3). The factors
that induce EMT in other cancers may also activate mesenchymal
features in gliomas (Fig. 1).
Furthermore, EMT is an important inducer of the cancer stem cell
phenotype (4). The mesenchymal
subtype of glioblastoma (GBM) typically expresses neural stem cell
markers and is associated with an aggressive phenotype (5–7). Glioma
cells that express stem cell markers are highly invasive and
resistant to chemotherapy and radiotherapy in vitro
(8–10)
and in the clinical setting (11).

Gliomas are classified according to their
histopathological features; these features allow clinicians to
distinguish between two cellular lineages (astrocytic and
oligodendrocytic) and four grades of malignancy (grades I to IV)
(12). The most malignant form of
grade IV is GBM, which originates from progenitor or stem cells in
the astrocytic lineage. Recent genotyping and expression profiling
analyses have demonstrated that GBMs may be categorized into four
subclasses dependent on their neural differentiation (5,6). The
proneural subtype is associated with a positive prognosis, whilst
the mesenchymal subtype is characterized by higher percentages of
cycling cells and neoangiogenesis, with a highly invasive nature
and poor prognosis (5,6). Furthermore, non-mesenchymal subtypes of
tumors typically acquire mesenchymal features at recurrence
(6). A shift towards the mesenchymal
subtype appears to be a common pattern in disease progression,
similar to cancer cells undergoing EMT in order to acquire a more
aggressive nature (13).

Paradoxically, migrating tumor cells are required to
lose the mesenchymal phenotype to establish a secondary tumor at
distant sites (1,2). This suggests that EMT is a reversible
process, and is most likely to be mediated by epigenetic
alterations that are induced by microenvironmental stimuli, rather
than as a result of genetic alterations (14,15).
Differentiated tumor cells change their phenotype through a dynamic
reprogramming process that is affected by a repair-associated
process or pathological stresses, such as hypoxic insults (16–20).
Acquisition of the stem cell phenotype may be closely associated
with epigenetic alterations that are induced by EMT. Although EMT
may be a common pattern in glioma progression, numerous therapeutic
interventions affect the occurrence and magnitude of EMT during the
clinical course of GBM (21–23). The present review discusses the
participation of EMT in GBM progression, and the resulting
acquisition of the stem-cell phenotype.

Classification of EMT

EMT is classified into three different subtypes
(1,2).
EMT type 1 is an essential mechanism required for the transitioning
of primitive epithelial cells in embryos into motile mesenchymal
cells, which are required for the gastrulation and migration of
neural crest cells (24). Certain
cells generated by EMT become secondary epithelial cells in
mesodermal and endodermal organs through a reverse event known as
mesenchymal-epithelial transition (MET). Thus, during
embryogenesis, type 1 EMT serves a critical role in generating
morphologically and functionally distinct cell types, including
mesenchymal and secondary epithelial cells, through the process of
MET (1,2).

EMT type 2 occurs in adults and is associated with
tissue regeneration, wound healing and organ fibrosis, in which
fibroblasts are formed in injured tissues. Organ fibrosis is
mediated by fibroblasts and inflammatory cells that secrete a
number of inflammatory signals alongside the components of a
complex extracellular matrix, consisting of elastin, collagens,
tenacin and laminins. The transition of epithelial cells into
fibroblasts occurring over a few days in culture is one line of
evidence for this type of EMT, and an active diversion to MET
occurs in the presence of bone morphogenic protein-7 (25). Cancer-associated fibroblasts in
primary epithelial tumor nodules have recently been demonstrated to
share certain genetic mutations with tumor cells, suggesting that
type 2 EMT emerges prior to the full onset of tumorigenesis
(26).

EMT type 3 is observed in subsets of cancer cells
undergoing a phenotypic conversion to increase migration, invasion
and metastasis. Certain studies have noted that transforming growth
factor (TGF)-β can induce EMT in epithelial cancer cells through
Smad or p38 mitogen-activated protein kinase/Ras homolog family
member A pathways (27–29). Activation of EMT programs, through
tumor microenvironment stimuli, has been proposed as the critical
mechanism for the acquisition of highly malignant phenotypes of
cancer cells (30). In type 3 EMT,
certain cancer cells with a transitioning mesenchymal phenotype
undergo MET to form metastatic tumor nodules at distant sites
(1,2).

EMT-inducing microenvironment

The genetic and epigenetic alterations that cancer
cells undergo render them sensitive to EMT-inducing signals. Highly
motile mesenchyme-like cancer cells are typically observed at the
invasive front, suggesting that dedifferentiating signals usually
originate from the tumor microenvironment (30). As aforementioned, the reversibility of
EMT suggests that epigenetic alterations, as a result of
environmental signals, generate highly aggressive tumor phenotypes
(31–34).

A hypoxic microenvironment is generally regarded as
a potent inducer of EMT in various types of epithelial cancer
(30,35). In gliomas, inflammatory processes, or
a hypoxic microenvironment within the tumor or neighboring normal
tissues, may result in the recruitment of circulating or
residential myeloid cells (including macrophages or microglia) into
the tumor stroma (34). These cells
release a number of growth factors, including TGF-β, epidermal
growth factor, platelet-derived growth factor and fibroblast growth
factor-2, which trigger alterations in the levels of transcription
factors required for the initiation of EMT, and also in numerous
proteases that increase invasiveness into the surrounding normal
brain (17,21,34,36). Thus,
glioma cells that are affected by the bystander myeloid cells and
such signaling molecules may undergo EMT in a hypoxic
microenvironment.

EMT-inducing signals in gliomas

Twist

Twist is a protein with a basic helix-loop-helix
structure and is transcriptionally active during cell
differentiation and lineage determination (37,38).
During the establishment of cancer metastases by EMT, Twist acts
independently of Snail to suppress E-cadherin and to upregulate
N-cadherin and fibronectin (38).
Using a brain slice culture and an orthotopic model of
xenotransplantation, it has been reported that Twist is upregulated
in malignant gliomas, and promotes glioma cell invasion through the
mesenchymal target gene Slug and the fibroblast activation protein,
independent of the cadherin switch (39,40). It
has also been demonstrated that the inhibition of Twist expression
results in a significant reduction in GBM stem cell sphere growth
and formation. Nagaishi et al (41) observed that the expression of Twist is
characteristic of mesenchymal areas of gliosarcomas, indicating
that EMT is involved in the formation of biphasic tumor
gliosarcoma.

Snail

Snail is a member of the SNAIL family of
transcriptional activators and is a primary suppressor of
E-cadherin expression (1,2,42). Snail
regulates a range of other EMT phenotypes, including the decreased
expression of various epithelial markers (occludins, claudins and
cytokeratin) and the increased expression of mesenchymal markers
(vitronectin and fibronectin) (43).
The transcriptional activity of Snail is predominantly regulated by
its subcellular localization. Phosphorylation of Snail results in
its exportation from the nucleus to the cytoplasm, leading to
inactivation of the protein as a transcription factor (42). TGF-β is secreted from mesenchymal
cells following irradiation and induces the nuclear localization of
Snail via Smad2/3 pathways (22).

Slug

Slug is another member of the SNAIL family of
transcriptional activators and serves an important role in
suppressing the epithelial phenotype in numerous cancer cells
(1,2,44). Slug is
closely associated with the increased migration and invasion of
malignant gliomas (45). A
multi-cancer mesenchymal transition signature of mRNA expression
levels from The Cancer Genome Atlas (TCGA) data has been
highlighted by strong expression of Slug and cluster of
differentiation (CD)44 (5,6).

ZEB

The zinc finger E-box-binding homeobox (ZEB)
proteins, ZEB1 and smad1-interacting protein-1 (also known as
ZEB2), are another family of noteworthy transcription factors that
are responsible for the mediation of EMT in numerous types of
cancer and glioma (1,2,46). ZEB
proteins bind to the promoter region of E-cadherin and suppress its
expression, resulting in the loss of cell-cell contact and
increased motility (47,48). Wang et al (46) observed that patients with GBM
containing high levels of ZEB2 demonstrated significantly earlier
recurrence with malignant transformation compared to those with low
levels of ZEB2. Connective tissue growth factor also renders glioma
cells highly invasive through the activation of nuclear factor-κB,
which subsequently initiates ZEB1 expression (49).

Wingless-related integration site
(WNT)/β-catenin

In multiple types of cancer, β-catenin is
sequestered in the cytoplasm by E-cadherin, with the translocation
of β-catenin into the nucleus following the downregulation of
E-cadherin being directly correlated with acquisition of the
mesenchymal phenotype (1,50,51).
Although the majority of GBMs do not express E-cadherin, nuclear
localization of β-catenin is primarily observed at the invasive
front of the tumor (52).
Furthermore, GBMs that express high levels of WNT/β-catenin are
correlated with significantly shorter patient survival times
(53). The WNT/β-catenin pathway is
an important stem cell maintenance pathway and is involved in
therapy resistance (54). GBM cells,
in which the WNT/β-catenin pathway is activated, trigger the
expression of a set of EMT activators, including Twist1, ZEB1,
Snail and Slug (55). Furthermore,
high expression levels of the WNT/β-catenin receptor, Frizzled-4,
promotes the expression of Snail and the acquirement of a
mesenchymal phenotype in GBM (56).

NOTCH

NOTCH is a cell surface receptor that serves an
important function in the development of numerous types of cells
and tissues (1). NOTCH signaling is a
primary inducer of EMT in a number of epithelial cancers, including
cancer of the lung, breast and pancreas (57). Fan et al (58) reported that inhibition of this
signaling pathway by γ-secretase inhibitors reduces CD133-positive
stem-like cells in GBMs. In addition to WNT/β-catenin, NOTCH is a
major regulator of glioma stem cells within their
microenvironments. NOTCH is also directly correlated with
phosphoinositide-3 kinase/Akt pathway activation (59–61).

CD44

CD44 is a hyaluronic acid receptor that interacts
with ligands such as collagens, osteopontin and matrix
metalloproteases (62,63). In addition to the standard isoform of
CD44 (CD44s), alternative splicing results in 11 other isoforms of
CD44 variants (CD44v2-v12) (64).
CD44s is a primary inducer of EMT in breast and colorectal cancer.
TCGA data indicates that GBMs with high levels of mRNA expression
of EMT-inducing signature molecules, including Slug and CD44, are
correlated with increased resistance to therapies and tumor
invasion (65). However, functional
data for CD44-mediated EMT in GBM have not been fully elucidated
(66).

MicroRNAs (miRs) that regulate EMT in
gliomas

miRs are small, 20–23-nucleotide non-coding RNAs
that serve as epigenetic regulators of gene expression through the
downregulation of target genes; this occurs through the binding of
miRs to regions of partial complementarity in the target gene
3′-untranslated regions (67). Each
miR has hundreds of target genes, and numerous genes are targeted
by multiple miRs, creating a highly complex gene expression
regulatory network (68). Control of
gene expression by miRs is one of the most important modulating
processes in cellular differentiation during normal embryogenesis
(69,70). A number of studies have demonstrated
that miRs may function as negative regulators of gene expression in
normal tissues and as tumor suppressors or oncogenes in various
tumors (67,70,71). In
several types of cancer, epigenetic regulation (involving miRs) is
a core mechanism of EMT modulation, and thus, reversible modulation
of the genes that mediate EMT is possible (72). The majority of miRs are negatively
correlated with tumorigenesis, tumor invasion and mesenchymal
changes in gliomas. Notably, the expression of miR-21, −34a, −128a,
−124 and −184 is correlated with the downregulation of mesenchymal
markers and decreased invasiveness. By contrast, a relatively small
number of miRs are oncogenic and may function as therapeutic
targets. The inhibition of a Dicer enzyme for a specific oncogenic
miR was recently indicated to block maturation of the miR and
suppress tumor invasion (73).
Furthermore, evidence is growing concerning the effect of miRs on
the progression and maintenance of glioma stem cells (67,71).

Radiation-induced EMT

Radiation therapy is a major modality of cancer
therapy and also serves a key role in the multimodal treatment of
GBM. However, irradiation that is sublethal to malignant glioma
cells consequently promotes cell migration and invasion through the
expression of TGF-β, epidermal growth factor, vascular endothelial
growth factor (VEGF) and the hepatocyte growth factor pathway
(74–76). Glioma cells that are resistant to
irradiation have a gene expression signature that is enriched in
the EMT pathway, leading to highly invasive recurrence patterns
(22,23,77,78). TCGA
data indicates a shift from a proneural to mesenchymal phenotype at
the time of tumor recurrence. Recently, Mahabir et al
(22) observed that two different
pathways are involved in the radiation-associated EMT induction in
malignant gliomas; TGF-β, derived from the mesenchymal cells in the
tumor environment, evokes the activation of Smad2/3, whilst
reactive oxygen species activate extracellular signal-regulated
kinase1/2, with each pathway leading to the nuclear localization of
Snail. Such data suggests that EMT serves a crucial role in the
acquisition of radiation resistance. Furthermore, emerging evidence
suggests that such a role for EMT in the generation of refractory
cancer cells is associated with an accumulation of stem cell
markers. The NOTCH pathway and WNT/β-catenin signaling are
important for stem cell maintenance and are associated with the
radiation resistance of GBM (78).

A further important aspect of the biological effects
of radiation therapy on GBM is the induction of hypoxia or
necrosis. Tissue hypoxia directly induces EMT and recruits myeloid
cells into tumor tissues (15–20).
Glioma cells, under a hypoxic microenvironment, and recruited
myeloid cells each secrete TGF-β, leading to the induction of
hypoxia-inducible factor-1α (HIF-1α), which subsequently promotes
the malignant progression of glioma cells (21,79).

Bevacizumab-induced EMT

VEGF is one of the most important factors
facilitating angiogenesis and resultant tumor growth in GBM.
Inhibiting the VEGF-VEGF receptor (VEGFR) signal transduction
pathway with anti-VEGF therapy (including the use of bevacizumab)
and VEGFR inhibitors (including sunitinib) is a promising strategy
in cancer therapy (80). Bevacizumab
is effective in prolonging progression-free survival (PFS) in newly
diagnosed GBM patients, but is not effective in prolonging overall
survival (81,82). During the early phases of bevacizumab
therapy, tumor oxygenation improves through the process of vascular
normalization (83). However, with
prolonged treatment with bevacizumab, in a similar manner to
radiation therapy, the tumor develops progressive hypoxia that
directly or indirectly promotes the mesenchymal phenotype (21,79,83).
Furthermore, hypoxia induces the release of HIF-1α from glioma
cells and subsequently attracts myeloid cells, including
macrophages and granulocytes, from bone marrow into the glioma
tissues (21,23). The recruited myeloid cells release
TGF-β, which then directly induces EMT in the glioma cells. Myeloid
cells also secrete multiple growth factors, including interleukin
(IL)-6, IL-10 and matrix metalloproteinases (15–19).
TGF-β, alongside VEGF, also recruits mesenchymal stem cells into
the glioma tissues, which contributes to the further malignant
progression of GBMs (83). By
contrast, VEGFR inhibitors lack the efficacy in PFS prolongation,
due to the induction of hypoxia in the early phase, without the
vascular normalization phase, or due to dose-limiting adverse
events. Similarly, anti-VEGF therapy is more effective than VEGFR
inhibitors in decreasing myeloid cell infiltration, which may
contribute to the efficacy of bevacizumab observed during early
phases.

Conclusion

Glioma cells undergoing EMT acquire the potential to
initiate metastasis and invasion. This process is highly affected
by the tumor microenvironment, particularly a hypoxic environment
or one involving the release of proinflammatory molecules from
recruited myeloid or mesenchymal stem cells. The evidence that type
I and II EMT occur during the normal physiological processes of
embryogenesis and wound healing in a relatively short time suggests
that epigenetic mechanisms are more crucial than genetic changes.
This notion is also supported by the evidence that migrating tumor
cells that have undergone EMT may also undergo MET to establish
metastatic tumor nodules. Therefore, tumor microenvironments are
emerging as a therapeutic target, particularly when in a hypoxic
state, which controls epigenetic alterations in tumor cells. The
microenvironmental modifier, bevacizumab, has recently been
developed; however, future clinical trials to maximize the efficacy
of anti-VEGF therapy are required, with the aim that such treatment
will normalize oxygen concentration and suppress the excessive
recruitment of myeloid and mesenchymal stem cells.