Cancer Therapy Vol 2, 195-200, 2004
Epithelial-mesenchymal transition and progression of oral
carcinomas
Review Article
Kazushi Imai*, Toshiyuki Okuse, Tadashige Chiba, Masako Morikawa, Kazuo
Sanada
Department
of Biochemistry, School of Dentistry at Tokyo, The Nippon Dental University,
Tokyo, Japan
__________________________________________________________________________________
*Correspondence: Kazushi Imai Department of Biochemistry School of Dentistry at Tokyo,
The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan;
Tel: 81.3.3261.8870; fax: 81.3.3261.8875; e-mail: KIMAI@Tokyo.ndu.ac.jp
Key words: EMT,
oral cancer, transcription factor
Abbreviations: E-cadherin, (CDH1); b-catenin, (CTNNB1); epithelial-mesenchymal
transition, (EMT); high mobility group A, (HMGA); LIM domain-binding protein,
(LDB); LIM-only protein, (LMO)
Received: 15 June 2004; Accepted: 01 July 2004;
electronically published: July 2004
Summary
Oral
carcinomas are devastating diseases with poor patient survival. This
unfortunate outcome could partly result from that carcinoma cells frequently
loose epithelial cell characteristics and gain mesenchymal cell-type features,
as referred to epithelial-mesenchymal transition (EMT). Induction of EMTs makes
carcinoma cells invasive and metastatic. Unveiling the mechanism for EMTs would
be a challenge for development of a novel strategy for patients suffered from
the disease. In this review, we will focus on transcriptional regulation of
carcinoma cell-EMTs, in addition to the WNT signaling pathway, based on our
recent findings.
I. Introduction
Metastasis of carcinoma cells requires several
steps, including detachment from the primary site, dedifferentiation, invasion
of the surrounding stroma and vessel walls, embolism, and stromal invasion and
proliferation in distant organs. With few exceptions, carcinomas are derived
from single somatic cells and their progeny. Carcinoma cells in the emerging
neoplastic clone accumulate within them a series of genetic and/or epigenetic
changes that lead to changes in gene activity and hence to altered phenotype
that are subjected to selection for tumor progression (Ponder, 2001). Loss of
epithelial morphology and acquisition of mesenchymal characteristics, often
referred to as the epithelial-mesenchymal transition (EMT), are typical for
carcinoma cells and predispose tumors to a more advanced state of progression
(Hay, 1995; Birchmeier et al, 1996; Thiery, 2002). The genetic
instability may trigger alterations in regulatory sequences of correct gene
expression and may accumulate EMTs in carcinomas from a standpoint of tumor
progression. Induction of EMTs in squamous carcinoma cells drives tumor
progression through enhancement of invasive and metastatic features (Oft et al,
2002; Grille et al, 2003). Identifying the mechanism(s) that is involved in
EMTs provides insights into understanding the pathway of tumor progression and
development of a novel strategy predicting tumor malignancy and may contribute
to long-term survival of patients (Thiery, 2002).
II. Activation of WNT signaling
pathway
Torrential
flooding of intracellular signaling establishes the biological status of
carcinoma cells, and their interaction makes difficult to understand the
primary pathway for tumor progression. Among numbers of the pathway, b-catenin (CTNNB1)-mediated WNT signaling is
stimulated in varieties of tumors. WNT was identified as an oncogenic gene
activated by chromosomal integration of Mouse Mammary Tumor Virus, and
constitutes a large gene family (19 members in human). Ligation of secreted WNT
molecules to cell surface receptors (frizzled and LDL receptor-related protein)
sparks signaling pathway (Seidensticker and Behrens, 2000). In this pathway,
WNT-frizzled binding abrogates kinase activity of glycogen synthase kinase 3-b, liberating CTNNB1 from degradation and
increasing the cytoplasmic-free CTNNB1 pool. An excess amount of CTNNB1
translocates into the nucleus and transcribes target genes. In the absence of
WNT, glycogen synthase kinase-3b forces CTNNB1
to degrade, resulting in a decrease of the free CTNNB1 pool (Seidensticker and
Behrens, 2000). WNT pathway directly activates expression of genes involved in
proliferation, invasion and EMTs of carcinoma cells (www.stanford.edu/~rnusse/wntwindow.html).
Therefore, it seems reasonable to suppose that the WNT pathway promotes
progression of tumors. In oral carcinomas, carcinoma cells express
keratinocyte-type WNTs (WNT 6 and 7A), but also miss-express fibroblast-type
(WNT3, 11 and 16) or other cell-type (WNT3A, 4, 7B and 14). Carcinoma cells
express WNT3 and activate the WNT pathway at the invasive front (Figure 1) (Uraguchi et al, 2004).
Analogous findings are reported in colorectal carcinomas (Kirchner and
Brablets, 2000; Brabletz et al, 2001; Takahashi et al, 2002). Since the WNT
pathway triggers EMTs (Eger et al, 2000), activation of WNT expression and
signaling in oral carcinoma cells may stimulate EMTs and progression of tumors.
However, activation of the WNT pathway also plays a pivotal role in
developmental and non-tumorgenic events (Okuse et al, manuscript submitted). In
these situations, the pathway will be terminated after completion of the
events. Thus, if the WNT pathway takes an indispensable part in tumorgenesis
and/or tumor progression, we should address a mechanism responsible for the
sustained expression of WNTs in carcinoma cells.
III. Miss-expression of
mesenchyme-specific transcription factors
It is
widely accepted that many of molecular pathways underlying tumorgenesis
represent aberrations of the normal developmental processes. In a majority of
tumors, transcription factors can be re-expressed that are derivatives of
embryonic cells in which the transcription factors are normally expressed
during embryogenesis. However, transcription factors can be expressed in
tumorgenic cells derived from those in which a particular transcription factors
are not normally expressed during development (Abate-Shen, 2002). PAX5 is
expressed in medulloblastoma, but not in the cerebellum from which this tumor
is derived (Kozmik, 1995). Miss-expression of transcription factors, as opposed
to the re-expression,
Figure 1.
Immunolocalization of WNT3 and CTNNB1 in oral carcinoma tissues. (A) WNT3 was localized to carcinoma
cells at the invasive front. (B)
Endothelial cells (small arrow), fibroblasts (arrowhead), and macrophage-like
cells (large arrow) adjacent to carcinoma cells were also positively stained. (C) Normal gingiva did not react to WNT3
antibody. (D, E) CTNNB1 stained
cell-cell junction of carcinoma cells. (F,
G) At the extremity of carcinoma invasion, CTNNB1 showed diffuse
cytoplasmic or nuclear staining. Bar = 100 mm (A, C-E) and 50 mm (B, F, G). Reproduced from Uraguchi et al, 2004 with kind permission from
Journal of Dental Research.
could provide phenotypic
alterations and accumulate the cellular trans-differentiation, especially EMTs,
in carcinomas. In this section, we provide evidence for miss-expression of
mesenchyme-specific transcriptional co-factors in oral carcinoma cells and
contribution for the pathology of diseases.
A. HMGA2
The high mobility group A (HMGA) family consists of
three members, HMGA2, HMGA1a and HMGA1b. A prominent feature of the HMGA family
is the three DNA-binding domains, termed AT-hooks, that bind to AT-rich DNA in
the minor groove. They have no transcriptional activity per se, but through
binding with other transcription factors, they organize the framework of the
nucleoprotein-DNA transcriptional complex and enhance the transcription of
several genes, which are specifically expressed in mesenchymal cells (Thanos
2002, Carey et al, 1988). Because
HMGA2 is predominantly expressed in undifferentiated mesenchymal cells during
development (Zhou et al, 1996), it has been hypothesized that inappropriate
activation of the HMGA2 gene in terminally differentiated mesenchymal cells
that initiate the tumorgenic pathway and leads to a mesenchymal tumor (Ashar et
al, 1995; Schoenmakers et al, 1995). However, very little is known about a role
of HMGA2 expression in carcinomas of epithelial origin. Quantitative analysis
of HMGA2 gene expression demonstrated that oral carcinomas ectopically express
the gene at levels 163.4 ± 90.4 (mean ± 1 S.D.)-fold greater than that of normal
counterparts. HMGA2 protein expression is identified in 73.8% of carcinomas and
predominantly seen in most carcinoma cells at the invasive front, where
carcinoma cells gain the characteristics of EMTs and facilitate tumor invasion
(Figure 2). In addition, HMGA2
protein expression is closely associated with tumor recurrence and patient
survival. This is highlighted by the fact that 100% of patients who died of
tumor recurrence express HMGA2 protein, and every HMGA2-expressing patient
without lymph node metastasis died of tumor recurrence. Furthermore, protein
expression is closely associated with the long-term patient survival rate
independent of other risk factors (Figure
3). Although survival of clinically metastasis-negative patients free from
disease recurrence is limited to 51.7%, 100% of HMGA2-negative patients survive
without tumor recurrence (Miyazawa et al, 2004). Treatment of clinically
metastasis-negative patients with chemotherapy or radiotherapy with neck
dissection is a controversial issue (Lippman and Hong, 2001). HMGA2 may be a
novel superior marker for tumor recurrence and examination of HMGA2 protein
expression by immunostaining on incisional biopsy specimens would predict tumor
aggressiveness and stratify patients into risk group.
B. LMO4 and LDB1
The LIM-only protein (LMO) carries two tandemly
repeat LIM zinc-binding domain, which acts as an adaptor for transcription
factors facilitating assembly of large transcriptional complexes (Breen et al,
1998; Sugihara et al, 1998). LMO4 gene is widely distributed in embryonic
tissues (Kenny et al, 1998; Sugihara et al, 1998), and involved in negative
regulation of breast carcinoma cell differentiation (Visvader et al, 2001).
LMO4 binds with a high affinity to the LIM domain-binding protein 1 (LDB1),
which binds to transcription factors and bridge a
Figure 2 Immunolocalization of HMGA2 in squamous cell carcinomas. A shows a
low-power view of HMGA2 staining. A high-power view of the staining at the
center and invasive front is shown in B
(depicted in inset b in A) and C (depicted in inset c in A), respectively. D, a carcinoma tissue section was reacted with nonimmune IgG
instead of anti-HMGA2 antibody as a negative control reaction. E, normal
epithelial cell of the gingiva was negatively stained. Bar, 250 (A), 50 (C and D), and 150 mm (E). Reproduced from Miyazawa et al, 2004 with kind
permission from Cancer Research.
Figure 3 Disease-specific survival in
oral carcinoma patients based on the expression of HMGA2. The graph summarizes
Kaplan-Meier survival analysis for patients with positive or negative HMGA2
staining. Statistically significant differences were examined between negative
and positive HMGA2 staining (P =
0.0006). Reproduced from Miyazawa et al, 2004 with kind
permission from Cancer Research.
Figure 4 Immunoreactivity of LMO4 and LDB1 at the primary site of oral
carcinomas. A positive direct correlation between LMO4 (horizontal line) and
LDB1 (vertical line) immunoreactivity was found by simple linear regression (r2 = 0.669, P < 0.01). Open circles, crossed and
shaded circles indicated well, moderately, and poorly differentiated
carcinomas, respectively. Reproduced from Mizunuma et
al, 2003 with kind permission from British Journal of Cancer.
unique bipartite DNA
sequence separated by about one helix turn from each other (Jurata et al, 1996;
Wadman et al, 1997). Both LMO and LDB proteins appear to have essential
functions in cell proliferation and lineage determination, and oncogenesis
(Jurata et al, 1998; Thaler et al, 2002). The LIM domain of LDB1 contributes to
the binding of transcription factors, including LIM-homeodomain, zinc-finger
and basic helix-loop-helix proteins (Jurata et al, 1996; Morcillo et al, 1997).
Formation of protein complexes synergistically activates the expression of
target genes (Jurata et al, 1998). However, in the presence of LMO protein, it
competes direct binding between LDB and the transcription factor (Rabbitts,
1998; Thaler et al, 2002). Miss-expression of LMOs by the chromosomal
translocation is observed in T-cell leukemia and inhibits differentiation of
neuronal cells (Thaler et al, 2002). We observed that normal keratinocyte and
oral carcinoma cells express LDB1, but LMO4 expression is only detected in
carcinoma cells. These proteins are predominantly expressed in carcinoma cells
at the invasive front, and upregulated in parallel with tumor dedifferentiation
(Figure 4) (Mizunuma et al, 2003).
Although biological consequences of LMO4 expression in oral carcinoma cells is
not certain, we are currently investigating the LMO4-binding transcription
factor and target genes.
IV. Downregulation of E-cadherin
expression
First step of carcinoma progression is dissociation
from cell-cell adhesion, which is mediated by E-cadherin (CDH1). An animal
model of pancreatic carcinoma demonstrated that a direct role of CDH1 in
adenoma-to-carcinoma conversion (Perl et al, 1998), indicating CDH1 as a tumor
suppressor gene. Disruption of CDH1-mediated cell-cell adhesion by anti-CDH1
antibodies induces EMTs of carcinoma cells (Imhof et al, 1983). Although loss
of tumor suppressor gene expression has been believed to result from the
classical KnudsonŐs two-hit hypothesis, emerging evidence indicate that somatic
mutation with loss of heterozygosity is extremely rare in sporadic cancer and
that epigenetic pathways are responsible for the lack of CDH1 in the majority
of sporadic carcinomas (Cheng et al, 2001; Graff et al, 2000). Recently,
transcriptional repressor, SNAIL, and promoter hypermethylation are considered
to be primary cause of CDH1 downregulation (Cano et al, 2000; Batlle et al,
2000; Graff et al, 2000). However, other CDH1 repressors, including SLUG, SIP1
and E12/47, are also known. We considered what epigenetic aberrations could
repress CDH1 in oral carcinoma cells. Unexpectedly, SNAIL expression was not
related to the CDH1 expression status. However, SIP1 expressing cells
negligibly expressed CDH1. Promoter hypermethylation was also predominantly
observed in CDH1-negative cells (Maeda et al, manuscript submitted). Synergistic
action and balance between SIP1 expression and promoter hypermethylation may be
critical determinant for the epigenetic loss of CDH1 during oral carcinoma
progression and plays a role in an induction of EMTs.
V.
Conclusion
In this review, we focused on molecular pathway for an
induction of EMTs in oral carcinoma cells. Transient alterations in cell
proliferation, differentiation, and migration activities initiated through
changes of microenvironments are observed in pathological and physiological conditions.
However, in the case of malignant tumors, it is well within the realm of
possibility that tumor cells have been destined for undergoing to EMTs by more
upstream critical aberrations. Aberrant expression of transcription factors or
activation of oncogenic signaling would be a candidate. Understanding the
molecular mechanism(s) for EMTs is not only an interesting issue cell
biologically but also may provide a novel strategy for cancer therapy.
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