POPESCU
IULIAN, PhD, MD,
The Radiobiology Department of
the Clinical Institute in Fundeni Bucharest
E-mail: popdociul@yahoo.com
ALINA HALPERN, PhD
"St. Stefan" Hospital, Bucharest
ABSTRACT
The
metastatic progression is responsible for most deaths from cancer in humans. For
achieving this it is necessary to convert the epithelial phenotype of cancer
cells into the mesenchymal phenotype that favours metastasis. This
transformation, which is transient, is called Epithelial-Mesenchymal Transition
(EMT).
The epithelial
cancerous cells are characterized by immobility, apical-basal polarity, strong junctions
of desmozomes, an interaction between the cell and extracellular matrix and an
increased expression of cell adhesion markers, where E-cadherin plays the main
role.
The mesenchymal
cell is mobile, multi-polar, fusiform aspect, does not make strong cell-cell
contacts, it can invade and express multiple markers, which confers these
properties.
The
TEM process is a complex mechanism involving changes in the expression,
distribution and /or function of several proteins. Among the main factors, we
distinguish:
CADHERINS.
They are a family of molecules that mediate the cell adhesion, intercellular
communication. The main member is E-cadherin,
which maintains the cell adhesion strength in combination with Integrins.
N-CADHERINE
contributes to mezenchymal transformation. The relation between the expression
of E-cadherine and N-cadherin – meaning the decrease in the E-cadherin
expression and increased expression of N-cadherin - is important in the TEM occurrence.
Beta catenin. It
is a cytoplasmic protein that interacts with E-cadherin. The relation between
them is essential for the induction and maintenance of the mesenchymal
phenotype. The neuro-endocrine lung tumors have an alteration of the E-cadherin
and Beta-Catenin expression in 90% of cases.
The TEM
occurrence is mainly related to the E-cadherin repression. Within the E-cadherin
repression mechanism the SNAIL genes mainly survene. SNAIL is a transcription
factor that represses the E-cadherin expression in epithelial cells, leading to
a predominantly fibroblastic phenotype. SNAIL may induce the expression of two
transcription factors, repressors of E-cadherin:
·
ZEB 1 that through
the inhibition of E-cadherin expression leads to the fibroblast phenotype acquisition,
it is a TEM activator, facilites metastasis and inhibits miR-200 (which is a strong
inducer of epithelial differentiation and stability).
·
ZEB 2 is another
repressor of E-cadherin (controlled by SNAIL) and inducer of TEM
SNAIL
is activated by NF-Kappa B in squamous carcinoma cells.
LOXL2
is an enzyme whose high expression leads to cancer dissemination and a low
survival rate. The SNAIL + LOXL2 common expression correlates in squamous forms
with malignant or local recurrence.
Within
tumors there are also mechanisms for keeping the epithelial cell adhesion and
differentiation. Thus:
Overexpression
of E-cadherin, beta-catenin sequestration in order to remain related to the
cell membrane and thus blocking its role as transcription factor, which is
important in delaying the occurrence of TEM. Further E-cadherin decreases the activity
of NF-Kappa B, which leads to an altered SNAIL transcription factor (usually
NF-Kappa B activates SNAIL), which further results in the decrease of ZEB
induction and other mesenchymal markers.
INTEGRINS.
They are trans-membrane receptor complexes, which transmit signals from outside
towards the cell inside in relation to cell adhesion, cell proliferation, cell
motility. Thus, the ALFAvBETA3 integrin increase leads to cell growth and
invasion, and the raised ALFAvBETA5 integrin increases the motility. In endothelial
cells, the interaction of ALFAvBETA3 and VEGFR2 integrins favors the tumor
angiogenesis, invasion and metastasis.
PERIOSTIN.
It is a mesenchymal specific gene whose protein is expressed in
osteoblast.Periostin targets the signaling coactivation of the ALFAvBETA5 integrins,
activates EGFR, raises the expression of Vimentin, Fibronectin and matrix
metalloproteinases, all contributing to the TEM development and metastasis.
PODOPLANIN.
This is a transmembrane protein. Podoplanin is involved in cell migration and
invasion. It is expressed in the marginal front area where tumor invasion
begins.
It
contributes to the cell dissemination, migration and invasion. It can induce
either unicellular invasion or group invasion, depending on the cellular
context. Podoplanin modulates the actin-cyto-skeleton by means of ERM proteins
(Eserine, Radixin, Moesin). Podoplanin modulates the RhoA activity regulation. The
podoplanin expression is increased in squamous forms, but is lacking in
adenocarcinoma. Podoplanin is a diagnosis and prognosis marker.
BRACHJURY
(T gene) It is a protein encoded by the T gene, being located in the 6q-27 area.
It is
a transcription factor, which mediates the EMT and invasion. Its overexpression
leads to the mesenchymal markers overregulation and epithelial markers
underregulation (mainly the repression of E-cadherin transcription), with a
concomitant cell migration and invasion.
The
tumor cells percentage with the Brachjury high expression increases with the
lung cancer stage. Its expression increases in lung cancer in stages II, III
and IV, but is absent in Ist stage and peritumoral normal tissue. In human
cancer cells Brahjury induces biochemical, morphological and functional changes
specific to EMT.
Its high
expression has diagnostic value and becomes therapeutic target.
INTER-LEUKIN-8
The induction of Epithelial-Mesenchymal Transition in cancer cells through the
overexpression of the Brachjury transcription factor is accompanied by an
increase in the secretion of cytokine, chemokine, angiogenesis factors. IL-8
has a particular role in cancer cells with the overexpression of the Brachjury
transcription factor. IL-8 plays a role in the acquisition and maintenance of
mesenchymal and invasive characteristics. IL-8 may potentiate tumor progression
by attracting other adjacent cells in epithelial-mesenchymal transition. IL-8
may be a therapeutic target against mesenchyme-like invasive cells.
TGF-BETA
(Transforming growth factor-beta). It is a proteine controlling the cell proliferation
and differentiation compared to epithelial cells it has an anti-proliferative
role. Cancer cells increase the production of TGF-beta. TGF-beta may contribute
to inducing the EMT with SMAD protein 3. The TGF-beta disorder leads to tumor development, modulates the cell
invasion, immune regulation, peritumoral changes.
The
TGF-beta effect is contextually depending
on the tumor development, difference between tissues.
HGF-MET
is another signaling pathway involved in the invasion increase. It induces two
genes involved in coagulation, whose overexpression leads to the
thrombo-hemorrhagic syndrome. Their role in cancer is to subregulate the
E-cadherin repressors: ZEB 1 and ZEB2. During TEM, 5 family members of miR-2000
are repressed by the action of TGF-beta.
Over or subregulation of various mi-RNA is essential for regulating the
epithelial phenotype, as well as the epithelial-mesenchymal transition (EMT) or
tumor progression.
Dicer is subregulated
in cancer and there is a connection between mi-RNA expression and Dicer.
Cancer-related Fibroblasts (CRC). They are activated and may have different
origins: in mesenchymal cancer stem cells from bone marrow through the local
fibroblast activation from cancer cells after EMT establishment. Although
different as origin, they co-exist. These cells detection within or around the
tumor helps us to decipher the origin of the multiple and various mesenchymal
cells.
INTRODUCTION
The
metastatic progression is responsible for most deaths from cancer in humans.
For
metastasis are required complex biological processes to detach cells, to send
them on vascular-lymphatic pathway in remote areas, where to survive and regain
the epithelial phenotype (1, 2, 3).
In
order to achieve this it is necessary a phenotypic conversion of the tumor
cells from the epithelial phenotype to mesenchymal phenotype and is related to
the metastatic potential acquisition (4, 5, 6, 7)
In
this process many molecules are involved in the cell motility, cell-cell
contact, interaction between cell and cell matrix (4, 6, 7). This transformation,
which is transitional from the epithelial phenotype to mesenchymal phenotype is
called epithelial mesenchymal transition
(EMT)
Physiological
Aspects: The EMT has been described for the first time during embryonic development,
when tissue modulation occurs in the cell migration to future organisms. BETTY
HAY (8, 9) highlighted the EMT in the gastrula process. It showed not only the mesenchymal
transformation, but also the fact that this event is transitional and may
return to the epithelial phenotype (1968-respectively-1995). In physiology, EMT
is essential in the mesoderm formation and neural tube formation (II) and wound
repair.
In pathological
terms, the EMT is an important step in tumor progression to metastasis and
wound repair. (5, 6, 7)
The epithelial
cell, which is stationary (not movable) is also characterized by apical-basal
polarity, strong junctions of desmosomes firm, interaction between cell and
extra cellular matrix (4, 6, 7) and with a high expression of cell adhesion
markers (such as E-cadherin (8, 9)
The mesenchymal
cell is movable, multi-polar, with fusiform aspect does, it does not make
strong cell-cell contacts, may invade and express markers such as: Vimentin,
Fibronectin, N-cadherin, Twist, Snail, Slug
There
is an opposite mechanism that is transition from the mesenchymal phenotype into
epithelial phenotype (EMT). The EMT is physiologically observed in the
development of various organs and pathologically in cancer metastasis. The role
in cancer is not well known. It is believed that it participates in stopping
and stabilizing distant metastases and allows the cancer cell to regain the
epithelial properties and integrate in the body where it has spread. The EMT
can be a therapeutic target for preventing metastasis (9)
THE
MOLECULAR BASIS OF MAIN FACTORS AND RESPECTIVE MECHANISMS
The EMT
is a complex mechanism that involves changes in the expression, distribution
and (or) function of several proteins (1-6,11,12,13)(5,10,11)
VIMENTIN.
It is a type III protein. Its increased expression leads to the replacement of
cyto-skeletal network and symbolizes the cell transformation into
mesenchyme-like cell (1-6,11) (5.10). Its expression increase is a main sign of
the proof of EMT existence, becoming a fibroblast-like phenotype which includes
the production dissemination and increase of the matrix-metallo-proteinases
that will lead to migration and metastatic potential (1:36)(4,12)
CADHERINE
Cadherines
are a family of molecules that mediate the cell adhesion (cell-cell), calcium
dependent and regulates the intercell communication. The gene that encodes
E-CADHERIN is CDH1. E-CADHERIN is necessary for the formation of adhesions
between the epithelial cells and maintains the epithelial properties (5:8,9)
(1,13,14). Thiery ((1-34) (4.6),
on measuring the cell adhesion force has observed that there are 2 cadherin
types: type 1, where cells adhere faster and stronger and type 2 cadherin,
which are migratory cells (CHU et al 2006) (15)
Moreover,
it has also been shown that the adhesion force of E-cadherin depends on the
interaction with Active skeleton cortical120 (glossary) and the presence of an activated
integrin, suggesting that Integrins and Cadherinele mutually control the
adhesion force (15)
Physiologically,
E-cadherin is essential in maintaining the cells epithelial integrity during
embryonic period and in repairing the adult tissue. The EMT main sign is the loss
of adherent junctions due to loss of E-CADHERINE expression.
N-CADHERINE
belongs to the classic group of cadherins and is found in neurons (wikipedia)
N-cadherin
contributes to mesenchymal transformation. In addition to this it has been
evidenced that N-cadherin overregulates MMP-9, which leads to tumor progression
(1:36,35) (7,12). In the EMT emergence it is the ratio of E-cadherin and N-cadherin
expression that matters.
The loss
of E-cadherin expression and (or) the gain of N-cadherin expression in the
tumor cell surface is frequent ans is correlated with the degree of
differentiation and aggressiveness (1:14) (4:16).
Thus
the N-cadherin gain and E-cadherin loss of expression of are frequently related
to metastasis (1:14,40,41). (4.16 * 17.18). In the EMT transition, epithelial
markers like E-cadherin are lost and mesenchymal markers such as N-cadherin and
Vimentin (7) (19) are gained.
In addition
podoplanin may lead to the increase of cell migration after losing E-cadherin
expression (7 martin Viller2006) (20., 21). Instead periostin does not change
E-cadherin şi N-cadherin expression (1)(4)
BETA-CATENIN
It is a cytoplasmic protein that interacts directly with E-cadherin.The complex
od E-cadherin-beta-catenin plays a crucial role in the cell-cell epithelial
adhesion and in maintaining the tissue architecture. The disturbance of this
complex in expression or function leads to the adhesion cellular loss and
further to tumor progressioni. Affecting the E-cadherin-beta-Catenin expression
is found in 90% of neuroendocrine lung cancers. Beta-catenin is located in the
3p21.3 area and has a tumor suppressor function rather than a oncogenic
function. Thus the Beta-catenin plays a
major role in cell signaling and cell growth facilitating (22.2325).
Cell
adhesion function depends on the integrity of the entire network E-cadherin,
BETA.CATENIN-actin (1CP). (24)
The
decrease of E-cadherin regulation leads to differentiation decrease and
increases tumor aggressiveness and frequency of metastasis.
Beta-catenin
interacts with E-cadherin in adhesion junctions in order to maintain epithelial
phenotype.
In
response to external signals, Beta-catenin is translocated from the extra
cellular membrane to cytoplasm, where it is spread and degraded, or may be
directly translocated into the nucleus, where it regulates the gene
transcription and contributes to the EMT induction.
Cell
density modulates the adhesion through E-cadherin beta-catenin turnover (Pocacici-Sorel
2003)(26). At low cell density, Beta-catenin is translocated into nucleus and
activates E-cadherin transcription repression by means of the SNAIL gene. This
is observed in the marginal tumor zone where it facilitates EMT and metastasis.
The common relation between E-cadherin and Beta-catenin is essential for the
induction and maintenance of mesenchymal phenotype (8 ) (26).
The
E-cadherin overexpression sequesters Beta-catenin and maintains it associated
with the cell membrane, preventing its role of important transcription factor
for EMT. E-cadherin helps in maintaining the epithelial phenotype by decreasing
the activity of NF-Kbeta. This decrease alters SNAIL activation and,
consequently, diminishes the ZEB gene induction and other mesenchymal markers (8-Garcia
de herreros)(27)
E-cadherin
REPRESSION is a crucial moment of the EMT, during the embryonic period and
adult tissues. First known repressor has been the SNAIL gene. Canno et al 2000
(28) has identified SNAIL as a direct transcriptional repressor of CDH1 and key
of events in the EMT process. Three repressor families intervene in this
processs of: Snail family, ZEB family and bHLH family. These repressors lead to
the repression of the epithelial cell polarity and cell division and thus forwards
cell survival (Peinado et al 2007) (29).
SNAIL
is a transcription factor that blocks (represses) the E-cadherin expression
from the epithelial tumor cell and fibroblast. SNAIL is involved in the E-cadherin
protein transport and its transcriptional repression (WU Mc CLAY) (30). SNAIL transfection
in epithelial cells leads to a predominantly fibroblastic phenotype. SNAIL
expressing cells have a phenotype where the contact between cells is decreased
diminuat. Also other epithelial markers such as cyto-keratin8 or MUC1 are
repressed.
SNAIL
induces the expression of mesenchymal markers such as fibronectin, LEF1
(glossary) and the repression factor of ZEB1 E-cadherin.
Both
SNAIL and ZEB1, which have similar expression in epithelial cells are in turn
induced by the ILK1 overexpression, a kinase, which leads to E-cadherin loss
and the acquisition of fibroblast phenotype (Sandra Guan?). ZEB1 (zinc finger
E-box binding home-BOX1) is an activator of EMT. It is a metastasis promoter.
ZEB1 also inhibits the expression of miR-200 family, which is a powerful
inducer of epithelial differentiation and stability (Ulrich Wellner)(31)
ZEB2 is
another repressor of E-cadherin. SNAIL1 controls ZEB2 action, which leads to
the increase of ZEB2 protein level (Garcia)(27). In the overregulation of ZEB2
and EMT induction there are also involved TGF-Beta, TNF Alfa, AKT and hypoxia.
The
induction of SNAIL and TWIST expression is performed by means of NFK-B and
leads to the formation of mesoderm. The SNAIL expression is activated by NFK-B
in squamous carcinoma cells with IGFR and AKT pathway (Julien S et al, L.Larue
Orsay, France) (32). Also it survenes in induction EMT, in mesothelial cells (Strippolin
R,Madrid Spania quote 26)
LOXL2
(Lyzyl oxidase homolog 2) is an enzyme, which in humans is encoded by the LOXL2
gene. The increased level of this enzyme is related to cancer dissemination and
leads to a low rate of survival. In squamous forms, SNAIL + LOXL2 expression is
correlated with malignancy or local recurrence (Peinnado 2005, 8).(33)
There
are also mechanisms for maintaining the differentiation and cell adhesion. E-cadherin
overexpression, beta-catenin sequestration leads to maintaining Beta-catenin related
to the cell membrane, preventing its transcription factor role, which is
important in the emergence of EMT. E-cadherin also helps in maintaining the
epithelial phenotype by decreasing the activity of NFKB (usually activates
SNAIL), which alters the SNAIL activity, which in turn decreases also the Zeb
induction and and other mesenchymal markers (Herrero)(27)
Besides
SNAIL genes have also been proven to be repressors other transcription factors:
E47,TWIST,ZEB,E2-2 (Battle.cano peinado 2007),(29)
INTEGRINS.
They
are hetero-dimer compounds of the transmembrane receptors that interact with
specific regions of the cell membrane protein and transmit signals from the
outside to the cell inside in relation to cell proliferation, cell adhesion and
motility. (1) (4)
Thus,
the ALFAvBETA3 integrin increase leads to cell growth, invasion (1:17,18)
(4,34,35), and the increased activity of the ALFAvBETA5 integrin increases
motility (1:19,20). (4,36,37).There is a mutual relation between integrins and
tyrosine-kinase receptors membrane (1:21,22,23). (4,38,39,40)
Thus,
the growth factors that activate tyrosine-kinase receptors, alter the
activities mediated by integrins in cell adhesion, spreading and migration.
Integrins
play an important role in activating both endothelial and tumor cells
(1:26,27)(41,42). Thus the connection between activating VEGF2 (FLK-1/KDR) and
ALFAvBETA3 integrins in endothelial cells is required for cell migration, cell
adhesion, while ALFAvBETA5 integrin is required for cell adhesion and cancer
cell motility (1:20,28,29). (4,37,43,44
The convergence
and signaling cascades from integrins and tyrosine-kinase receptors are
essential for endothelial cells and tumor cells in order to induce tumor
angiogenesis, invasion, metastasis. Thus, in endothelial cells, the interaction
of ALFAvBETA3 and VEGFR2 integrins is essential for VEGF to induce angiogenesis
(1:46,47)(4,45,46,)
PERIOSTIN
It is
a secretory protein (mesenchymal-specific gene) that is normally expressed by
osteoblast (Gillan et al) (1:30) (4,47). The authors found that periostin forwards
adhesion and enhances the cancer cell motility by fixing on ALFAvBETA 3 and
ALFAvBETA 5 integrins.
Also,
the increased expression of perostin correlates in human cancer with tumor
angiogenesis and metastasis (1:31,32, 33)(4,48,49,50). Administration by the
authors (1) of periostin-to-experiment 293T cells resulted in the fact that
periostin promote TEM. When this experiment was an increase of Vimentin,
becoming a fibroblast-like phenotype, which includes the production
dissemination and increase of Matrix-metalloproteinase (MMP). It further appears
the migration and metastatic potential.
In order
to obtain the aggressive response after administration of periostin is also
required the interaction with ALFAvBETA5 integrins and the activation of EGFR basal
level and tyrosine-kinase receptors (1:45). Clinically, the Periostin high
concentration correlates with a poor prognosis and short survival. Periostin
may be a marker for the metastases presence. The high production of periostin
may induce the malignant transformation of mesenchymal phenotype.
Periostin-
In summary, targets: co-activation of ALFAvBETA 5 integrins signaling, EGFR
activation, increased expression of Vimentin, Fibronectin, MMP all contributing
to EMT developments and metastasis.
Basically
in patients with high expression of EGFR and Periostin, may be administered tyrosine-kinase
inhibitors to control metastasis. (1:33,49,50)
PODOPLANIN
Podoplanin
is a mucin-like transmembrane glycoprotein, of small dimension. It is normally
expressed in the lung, heart, kidney (renal breitender podocytes 1999, martin
villa2005) on alveolar cell surface type 1 in rats (Rishi1991). It is expressed
in lymphoid and lympho-angiogenesis (breitender1999) and mesothelial cells
(ordonnez2006).
The physiological
function is not well known. From the experimental data appears to have a role
in development and tissue repair and carcinogenesis.
Podoplanin
is involved in cell migration and invasion both in vitro and in vivo (7).
Until
recently tumors have been considered anaplastic cell masses, without tissue-type
organization. But now it has been noticed a difference between cells molecular
expression from the invasive front of the tumor, which is different from inside
the tumor.
Malignant
tumors have the principal sign of malignant cell invasion in peritumoral tissue
and normal tissue architecture destruction. Morphologically we distinguish two
aspects of unicellular invasion and collective invasion (7).
In
the invasive frontal area, in molecular terms it is observed:
Nuclear
localization of beta-catenin, beta 1 integrin overregulation, the presence of LI1
cell adhesion molecule (wikipedia)( brablet2001,hegerfeld 2002,gavert2005)
In
human locations of the squamous carcinoma (including the pulmonary form)
Podoplanin is expressed on a cell layer at the edge of invasive tumor (Wicki
2006).
Podoplanin
expression at the edge of invasive front is influenced by : EGF,FGF2 (basic
fibroblast growth factor 2), TNF alfa,de bradykinin
fibroblastts(schull1997,Nicki2006)
Podoplanin
expression can be modulated by peritumoral tissue. The Podoplanin regulation
mechanism is not well known.
Podoplanin
facilitates the cell dissemination, migration and invasion. Podoplanin
expression leads to increased dissemination produced by fibronectin (Wicki
2006) and can be blocked by Beta 1antibodies integrin (wicki2001).
Cell
invasion expressing Podoplanin is related to the MMP activity, and may be
inhibited by TIMP2, an MMP inhibitor (Glossary).
Podoplanin
may lead to single-cell migration increase after the loss of E-cadherin
expression (Martin viller2000!). After Wicki (7) Podoplanin is able to induce
invasion in two situations- unicellular or collective group. It is not known on
what criterion this decision is made. It probably depends on the cellular
context. Other works are further required.
PODOPLANIN
modulates the cyto-skeleton,
Membrane
proteins are linked to AKTIN-CYTOSKELETON by means of (via)
ERM(EZRIN;RADIXIN;MOESIN) proteins. Ezrin mediates the filopodia formation
(glossary) and induce metastasis (Yu et al2004). Filopodia are cytoplasmic
extensions of the migratory cells (wikipedia). Podoplanin is physically linked
to Ezrin (SCHOLL1999). Podoplanin overexpression leads to the marked increase
of Ezrin phosphorylation and other ERM proteins, but without affecting the
level of Ezrin and MOESIN proteins (martin viller2006, Wicki 2006). ERM
proteins phosphorylation can bind podoplanin expression to the changes in
actincytoskeletal rearrangements (7).
Apart
from the ERM protein function, the RhoA activity (glossary) is modeled by
podoplanin. Podoplanin sub-regulates RhoA for MLF 7 cell experimentation, which
shows a high level of RhoA. Instead, in the MDLK cells, which show a low RhoA activity,
podoplanin increases the RhoA activity.
This
would be due to the different structure of the cyto-skeleton in the 2 cell
types (Martin Viller 2007). Apparently the RhoA activity regulation is involved
in the migratory phenomenon observed in cancer cells expressing podoplanin
(Martin Viller2000, Wicki 2006). Podoplanin is a tool for the cancer diagnosis
and its therapy. Through histochemical methods has increased the possibility
that Podoplanin expression be used as diagnostic marker and prognosis. Podoplanin
expression is detected in squamous cancer, brain tumors and germinal neoplasm.
These cancer cells tend to migrate collectively.
In
contrast, podoplanin is not found in adenocarcinoma (lung, colon, prostate).
Podoplanin
is overregulated in lung squamous form (Wicki, 2004). Also, it is expressed in
93% of mesothelioma (ordonnez2006).
Multiple
experiments claim that tumor cells can solitarily invade after losing intercell
adhesion or migrates in group without losing the cell contact. In squamous
forms, podoplanin is involved in migration in group, irrespective of EMT
(Wicki2006). However it was noted that in case of MDK cells and in oral
squamous cancer podoplanin may also facilitate unicellular invasion,
contributing to cell motility mediated by EMT (Martin viller2006).
In
these two migration pathways different factors survene. In collective migration
also intervene the members of TGF-Beta FAMILY (for example, NODALI), FGF2, Wnt
signaling, E-cadherin adhesion molecules şi eomesodermin (glossary).
In
unicellular the embryonic migration intervene TGFBeta other members (e.g.BMP),
FGF and Wnt (Locasoo,Netto 2001).
So
there are many factors able to induce the migration and invasion, activating
one of the two forms.
Induction
of podoplanin expression is the result of multiple adjustments of several
intracellular signaling pathways as modulations of the GTP actions, RhoA
family, ERM phosphorylation, Aktin-cytoskeletal rearrangements that ultimately
increase cell migration and invasion. The fact that Podoplanin is not found in
adenocarcinoma, but they show a collective migration, suggests that there may
be other ways for a migration independent of Podoplanin. Deeper studies of the
cellular pathways that lead to invasion helps us to use more effective
therapeutic strategies.
BRACHJURY
Brachjury
is a protein, which in humans is encoded by the T.Brachjury gene, is a
transcription factor complex within the T-box genes. It is found in animals
which have bilaterality. Brachjury has the symbol of T gene and name and is
located in the 6q-27 area.
It is
a diagnostic marker for chordoma. It is overexpressed in many tumor types
(5:22,23,24). Mediates the EMT and furthers the invasion (Wikipedia). Brachjury
(T-box transcription factor) is required for the normal mesoderm formation
(5:26,27,31, 32,33).
Brachjury
overexpression lead to over regulation of epithelial markers and mesenchymal
markers subreglarea with concomitant cell migration and invasion.
Brachjury
overexpression leads to transcriptional repression of E-cadherin, an effect
partly mediated by SLUG (transcriptional repressor). If obtained the silenced
Brachjury expression; in brachjury positive cells is produced the mesenchymal
markers subregulation and over regulation of epithelial markers, while
migration loss and lung metastasis formation.
The
percentage of tumor cells with Brachjury increased expression increases along
with the disease stage. Brachjury becomes an attractive candidate-target for
antitumor therapy (5).
Brachjury
overexpression in human cancer cells induces a series of biochemical,
morphological and functional changes characteristic for EMT. The fact that
alongside with brachjury other 2 SLUG and SNAIL transciptase factors intervene
shows that a variety of transcription factors take part in the changes occuring
in EMT (5:30). The brachjury overexpression leads to silencing the E-cadherin
expression, which is partly mediated by SLUG, while SNAIL does not take part
(5).
Brachjury
contributes to the acquisition by neoplastic cells of the mesenchymal phenotype
that includes mobility and invasion.
Brachjury
inhibition leads to inability to invade the extracelular matrix. Brachjury
inhibition is also accompanied by the expression reduction in genes encoding
MMP2 and MMP24. These two proteins are over-expressed in tumors and play a role
in cancer progression (5:37,38,39).
Brachjury
expression reduction leads to morphological and biochemical changes indicating the
reverse transition from the mesenchymal phenotype in epithelial phenotype (5 data
from experiment on H460 cells). In the experiment has been shown that brachjury
attenuates H460 cell proliferation in vitro suggesting that brachjury
intervenes in cell cycle progression, probably at the time of G1-S transition
by suppressing Cyclin D1 expression and inhibition of CDK compounds (5:42).
The
increased expression of brachjury has been present in samples of lung cancer in
stages II, III and IV, but was absent in patients with stage 1 and in normal
tissue without lung metastases (5).
The brachjury
increased expression correlates with the decrease of disease absence time
(progression free survival). Thus brachjury becomes a therapeutic target,
especially in advanced stages (5). Brachjury is an attractive therapeutic
target, due to multiple events that converge to increase the expression. If we target
the positive tumor cells for brachjury on the immunotherapy pathway with T cell
specific to brachjury we will make a more comprehensive immuno-therapy than the
targeted treatment of the signaling pathways (5:25).
In
conclusion, the data indicate that brachjury is able to induce morphological,
biochemical and functional changes characteristic to EMT in human cancer cells.
Its
overexpression in lung carcinomas, along with its functional capabilities show
that a gene is associated with tumor and may become a therapeutic target.
INTER-LEUKIN-8
The EMT
is known to induce tumor cell motility and invasiveness, leading to metastasis.
Although many studies have investigated the events that initiate this change (passing),
it was not elucidated the way of action of soluble mediators released by tumor
cells suffering transition and the impact had by this phenotypic change on peritumoral
region remodeling.
The
EMT induction in human cancer cells via the overexpression of the Brachjury
transcription factor is associated with a secretion increase of multiple
cytokines, chemokines, angiogenesis factors, in particular the induction of
IL-8/IL-8R axis (6).
The authors
data (6) show the essential role of IL-8 signaling for its acquisition and (or)
maintenance of mesenchymal and invasive aspects (characteristics) of tumor
cells with the overexpression of brachjury and show that the IL-8 secreted by
tumor cells and that undergoes the EMT transition may potentiate the tumor
progression by attracting also other adjacent cells in the transition EMT.
Globally,
their results show us the potential role of EMT in modulating peritumoral
tissue through the secretion of several soluble mediators and suggests that blocking
the IL-8 signaling may be a therapeutic target, very efficient against invasive
tumor mesenchyme-like cells.
TGF-BETA(transforming
growth factor-beta)
TGF-BETA
is a protein that controls cell proliferation, differentiation. It has -
compared to normal epithelial cells - an antiproliferative role and occurs in
the early stages of oncogenesis.
Cancer
cells increase the TGF-beta production.
TGF- beta mutations cause immunosuppression
and angiogenesis and cancer becomes aggressive.(Wikipedia)
TGF-BETA
may induce EMT, it is now well known the subsequent changes cascade (,6.7). Classically,
the TGF-BETA signaling requires:
-fixing
TGF-BETA to TGF-BETA receptor2
-transphosphorylation
of the type 1 receptor of the TGF
- later
the SMAD2 and SMAD3phosphorylation
It is
then translocated into the nucleus where it interacts as a coactivator,
corepresor of the transcription factor to suppress the epithelial gene
expression and promote the mesenchymal proteins.
To
these are added: activating the kinases ERK MAP,Rho-GTP-ases and
PI3-KINASE-AKT.
And
thus is induced the EMT (8,,10)
Besided
it has been noticed that SMAD3 (Wikipedia) is required for TGF-BETA to induce
the EMT. The SMAD3 protein level models the celular response to
TGF-BETA(HILL-LondonCITAT 8). There is also the relatioin between TGF-BETA and
other signaling pathways.A.OSTMAN showed the connection with the PDGF(glossary)
in zona peritumoral area and EMT induction TEM(ostman,Heldin2007).
TGF-BETA
also regulates BMP (bone morphogenetic proteins). BMP may induce Tem or EMT
depending on the context(JIAN XU et al).
The
TGF-BETA disorder leads to tumor growth, it may modulate the cell invasion, immune
regulation, peritumoral changes. The TGF-BETA response occurrence is very
contextual depending on tumor developments, differences among tissues.
The
clinical relevance of TGF-beta role in cancer is growing and helps us
understanding the complexity of cancer evolution and has therapeutic potential (11,12).
Another
signaling pathway involved in the invasion increase is HGF-MET. It induces two PAI-1
genes of coagulation (plasminogen activator-inhibitor-tip1) and Cyclo-oxigenase
2, whose overexpression induces a thrombo-hemorrhagic syndrome (P.COMOGLIOcitat8+boccacioand
p Comoglio)
MICRO-ARN
and TEM
Micro-RNA
are short-non-coding- Rna and are
involved in some development processes such as differentiation and
proliferation. Recently we have seen that are involved in cancer. The genes
encoding miRNA are either tumor-supressor genes or oncogenes (wienholds and
Plasterk2005). Genetics analysis showed an altered expression of miRNA in many
human cancers.
The miRNA
expression is subregulated in cancer and damaging the miRNA processing
increases the cell proliferation. Disturbance in miRNA expression results in
part from deficiencies in the miRNA development process(Courtois)
It
was shown that 5 members of the mir-2000 family are repressed during EMT by the
TGF-Beta action (gondall-australiacitat 8). It seems that their normal function
is to subregulate the repressors of E-cadherin (ZEB 1 and SIP1ZEB2).
The
data suggest that overregulation or subregulation of different miRNA is
fundamental for regulating the epithelial phenotype, as well as of EMT or tumor
progression (MA et al2007)
DICER
Dicer
is a highly conserved protein and is found in almost all eukariotic organisms.
This protein - encoded by the gene with the same name, works as a type III RNA
endonuclease and is required for the i-RNA pathways (interference) and mi-RNA
pathways .The Dicer gene is located in the 14q32.13 area. Her abnormalities
were observed in breast cancer, ovarian melanom. The Dicer mutation was
observed in the familiar pleural and pulmonary blastoma in pediatrics (Hill et
al2009). The Dicer expression alteration is observed in cancer.Merrit et al2008
have shown a decrease in Dicer expression in advanced adenocarcinomas and
ovarian cancer.
The Dicer
expression (Courtois) is associated with EMT and mesenchymal phenotype i.e. with
an expression subregulation in mesenchymal phenotype (Grelier et al 2009). Thus
the DICER expression is repressed inEMT. Dicer expression decrease leads to the
increased expression of ZEB1 and ZEB2 proteins and induces EMT.
There
is a correlation between the decrease of miRNA expression and Dicer expression reduction.
The p53
tumor-suppressor gene may be involved in Dicer subregulation and EMT induction.
It
seems that p53, p63, p73 regulates the major components of miRNA and DICER
pathways processing. There is a link between p63 and Dicer, regulating each
other. p53, p63 and p73 regulates miRNA and inhibits tumorigenesis, EMT,
metastasis and proliferation of cancer stem cells.(Courtois)
CANCER-RELATED
FIBROBLASTS (CAF)
Ostman
studied cancer-related fibroblasts and their interaction between malignant
cells and stroms. CAF are activated myo-fibroblasts and may originate in the
bone marrow, so-called mesenchymal cancer stem cells (Karnoud 2007), or are
generated through the local fibroblast activation or may have origin in cancer
cells after EMT (Brablet2005).
These
CAF cells differ in origin- they coexist. It is crucial to trace all these CAF
cells within or around the tumor, to decipher the origin of multiple and
various mesenchymal cells.
Thus
we will be able to know better tumor biology and we can foresee the future
anti-invasive medicines.
COMPARATIVE DATA
BETWEEN THE Epithelial phenotype and mesenchymal phenotype
·
Epithelial
phenotype.
- The epithelial cell is stationary, shows
an apical-basal polarity, strong
junctions of the desmozomes, interaction between cells and cellular matrix and
with an expression of cell adhesion markers such as E-Cadherin.
- E-cadherin maintains the epithelial
phenotype by decreasing the activity of NF-k BETA. Thus is reduced the SNAIL
suppressor activity, which in turn leads to the decrease of ZEB gene expression
and other mesenchymal markers.
- Beta-Catenin has a crucial role in
inter-cellular adhesion. The altered expression of E-cadherin and BETA-Catenin
is found in 90% of lung, neuro-endocrine cancers.
- Other epithelial markers are cytokeratine
8 and MUC-1.
- Micro-RNA are involved in the process of
development, differentiation, proliferation. MiR-200 family is a strong inducer
of the epithelial differentiation and stability. Their normal function is to
subregulate the suppressors of E-cadherin (ZEB 1 and ZEB 2). Overregulation and
subregulation of various mi-RNA is essential for regulating the epithelial
phenotype.
- DICER is a protein encoded by the gene with
the same name and highly conserved in all eucaryotic cells. There is a
correlation between mi- RNA and DICER gene. Together they contribute to the
inhibition of tumorigenesis, TEM, metastasis and proliferation of cancer stem
cells.
·
Mesenchymal
Phenotype.
-
The
cell is mobile, multipolar, has a fusiform appearance, does not make cell-cell
contacts, can invade, expresses markers such as Vimentin, N-Cadherin,
Fibronectin, Snail, Slug. Zeb1, Zeb2 etc.
-
N-cadherin. The increase of its
expression correlates with the degree of differentiation and aggressiveness.
-
The altered
expression of Beta-Catenin leads to Snail activation, which represses the
expression of E-cadherin.
-
Snail
is a transcription factor that represses the expression of E-cadherin, which
leads to a predominantly fibroblastic phenotype.
-
Snail
induces the expression of mesenchymal markers such as fibronectin, Zeb1.
-
Zeb1
is aTEM activator and promoter of metastasis. inhibits the expression of the
mi-R200 family, which is a potent inducer of the epithelial differentiation and
stability.
-
Zeb2
is an E-Cadherinei repressor.
-
In
EMT induction are also involved TGF-BETA,TNF-ALFA, AKT and hipoxia.
-
LOXL2.
Its increased level is connected to
cancer dissemination.
-
Integrins
lead to cell increase and invasion (alpha v beta3) and an increase in motility
(alpha v beta5). The link between integrins and receptor tyrosine-kinase induces
tumor angiogenesis.
-
Periostin
enhances the cancer cell motility through integrins. It is a marker of
metastasis.
-
Podoplanin
is involved in cell migration and invasion. Its overexpression is detected in
squamous cancer, but not in adenocarcinoma.
-
Brachjury
is encoded by T-gene. Is overexpressed in tumors, mediates EMT and promotes
invasion. It is a suppressor of E-Cdherinei. Its expression increases along
with the disease stage (II, III, IV),
-
But
it is not observed in stage 1. Is a treatment target through immuno-therapy.
-
Il-8 is
secreted by tumor cells, which undergoes the EMT transition, may potentiate the
tumor progression; shapes the peritumoral tissue by secreting soluble mediators.
-
TGF-BETA
has high values in cancer cell. The TGF-beta
mutations lead to immuno-suppression, angiogenesis, increasing the aggression. The
TGF- beta disorder is leading to
tumor development, modulates invasion, immune regulation, peritumoral changes.
-
Mi-RNA
expression subregulation in cancer leads to cell proliferation. They are
repressed by the TGF-BETA action.
-
The
DICER expression subregulation is related to EMT and mesenchymal phenotype. The
DICER expression subregulation leads to increasing the expression of the Zeb1
şi ZEB2 proteins and induces EMT.
Epithelium has strong structures.
A single layer or multiple layers and a glandular appearance. Such a structure
has an apical dorsal polarity, a transepithelial barrier with adherent or
separate junctions and an epithelium adhesion, cell-cell type, mediated by
adherent junctions and extra-cellular matrix similar to basement membrane.
The EMT is a transdifferentiation
program allowing the epithelial cell to lose their adherent junctions, to lose
the apical-dorsal polarity, to gain migration properties and also gain
resistance to apoptosis. Mesenchymal cells become capable to degrade the cell
membrane and migrate from the epithelium of origin. This complex activity is
driven by molecular processes such as the loss of E-cadherin expression (a cell
surface protein), change in the expression of transcription factors, the
expression of several new proteins like Vimentin and proteases as well as the changes
in RNA and Dicer expression.
There are 3 types of EMT
Type 1 This type refers to the
EMT during placenta implantation. The process is initiated by a specific
trophoblast subtype, that is Extra villous cytotrophoblast. Further, next stage
of the EMT process is gastrulation. EMT plays a major role in mesoderm formation and further
in organs developments.
Type 2. This refers to EMT in the
tissue repair and fibrosis processes.
Type 3 It concerns tumorigenesis
in cancer progression and metastasis process.
In embryonic developments, the regulation
of EMT and EMT inducers the process is orderly and has an end for the organ or
body functioning. The EMT in cancer is a process in which regulation is disordered
and unpredictable. The reactivation of embryonic process is related to a severe
prognosis.
The technical scientific
conquests helps us understand the EMT, which is found in many organ
developments and highlights the association of cellular processes with molecular
ones. These data highlight the behavior of migratory cells both in
physiological conditions (embryonic development, wound healing) and in pathological
conditions and confirms that EMT is an important step in the metastatic cascade
of carcinomas. The markers characterization will help to identify the nature
and origin of all mesenchymal cells found in the tumor, stroma or near the
tumor.
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