Download DSG3 in the carcinogenesis of head neck cancer

DSG3 in the carcinogenesis of head neck cancer
Yin-Ju Chen1,. Ann-Joy Cheng2
1
Graduate Institute of Basic Medical Science, Chang Gung University, 2School of Medical Technology, Chang
Gung University, Taoyuan 333, Taiwan
Abstract
To identify genes that could potentially serve as molecular therapeutic markers for human
head and neck cancer (HNC), we employed differential display analysis to compare the gene
expression profiles between HNC and histopathologically normal epithelial tissues. One of
identify gene DSG3 is over-expression in HNC. Inhibition of DSG3 by RNAi significantly
reduced cell growth and colony formation to 57% ~ 21% in three HNC cell lines. Use of an
in vitro wound healing and Matrigel invasion assays, we found that cell migration and
invasive ability were also inhibited to 30% ~ 48%. These results demonstrate that DSG3
plays important role in the regulation of cell growth and invasion. Since DSG3 is associated
with plakoglobin in desmosome, we hypothesized that DSG3 knockdown, through Wnt
signal pathway, increases the competitive binding of plakoglobin with β-catenin resulting to
the reduction of Tcf/Lcf transcriptional activity. From immuno- precipitation and
immunofluorescent staining analysis, we found that, DSG3 knockdown disrupted the
interaction with plakoglobin, and increased the translocation of plakoglobin from cytoplasm
to nuclear. By luciferase reporter assay, DSG3 knockdown significantly reduced Tcf/Lef
transcriptional activity and subsequently suppressed the expressions of Tcf/Lef downstream
target genes, including c-myc, cycline D1 and MMP7 which involved cell proliferation and
invasion regulations. Moreover, an in vivo xenograft study showed that administration of
DSG3-RNAi plasmid inhibited tumor growth by 83% for 2 months in BALB/C nude mice
(P< 0.001). In conclusion, DSG3 is over-expression in HNC. Suppression of DSG3
expression can intervene plakoglobin functions, leading to the reduction of carcinogenesis in
HNC.
Introduction
Potential role of desmosome and DSG3 in cellular function
Desmoglein 3 (DSG3) is one of the components in desmosome. Desmosomes are button
like points of intercellular contact which couple cytoskeletal elements to the plasma
membrane at cell-cell or cell-substrate adhesions. Whereas the related adherens junctions
associate with microfilaments at cell-cell interfaces, desmosomes are tailored to anchor
stress-bearing intermediate filaments at sites of strong intercellular adhesion (Garrod et al.,
2002; Green & Gaudry, 2000; Yin & Green, 2004). The resulting supracellular scaffolding
plays a key role in providing mechanical integrity to tissues. These cellular rivets are
dynamic structures subject to transcriptional and post-translational regulatory signals, and
also participate in cell morphogenesis, differentiation and proliferation.
Desmosomes comprise proteins from at least three distinct gene families: cadherins,
armadillo proteins, and plakins. The desmosomal cadherins are further divided into
desmogleins (DSG1-3) and desmocollins (DSC1-3). The armadillo proteins include
plakoglobin, plakophilins (PKP1-3), and p0071. The plakin family proteins include
desmoplakins, plectin, and the cell envelope proteins envoplakin and periplakin.
In vitro binding assays, along with a high-resolution map of the desmosome obtained by
electron microscopy, support an arrangement of proteins similar to that demonstrated for
adherens junctions. Members of both subfamilies are single-pass transmembrane
glycoproteins that mediate Ca2+-dependent cell-cell adhesion. A cadherin tail binds to an
armadillo family member (β-catenin and plakoglobin in adherens junctions or plakoglobin
in desmosomes), which in turn associates with a cytoskeletal linking protein (β-catenin in
adherens junctions and desmoplakin in desmosomes) (Figure 1). Since DSG3 participate in
the desmosome adhesion, belonging to non-classical cadherin family, it is possible that DSG3
like classical cadherin, also mediated cancer cellular function..
Figure 1. The junctional complex of desmosome. (A) Electron micrography. (B) Molecular
model of adheren junction. (C) Molecular model of the desmosomes.
(B)
(A)
(C)
From above literature reviews, I compared and summarized characteristics of the cellular
junction proteins of the E-cadherin, N-cadherin and DSG3 and listed in the Table 1. Currently,
most studies are focus on the functions of E-cadherin and N-cadherin in cancer cell migration
but little on the function of DGS3 during cancer formaiton. The function of this non-classical
cadherins, DSG3 in tumor progression is unknown.
Table 1. Characteristics and Comparison of three cadherin molecules
E-cadherin
Cadherin
Junction association
Main location
classical cadherin
adherin junction
epithelia
N-cadherin
classical cadherin
adherin junction
α-,β-,γ-catenin
non-classical cadherin
desmosome
Neurons, heart,skeletal
muscle, fibroblast
Interaction molecules
DSG3
α-,β-,γ-catenin
skin
stratified epithelia
γ-catenin (plakoglobin)
plakophilins desmoplakins
Cancer cell migration
Signaling pathway
invasion suppressor
RTK, WNT
invasion promoter
FGFR, MAPK
unknown
unknown
Desmosome molecules, β-catenin and plakoglobin as signal transducers
In addition to maintaining cell-cell adhesion, the downstream interacted protein of
E-cadherin, β-catenin also regulates signaling. When cell maintains cell-cell adhesion, all the
β-catenin molecules are sequestered with E-cadherin, therefore, there is no more free
β-catenin in the cytoplasm to regulate TCF/LEF1 family of transcription factors and their
target genes. If cell loss E-cadherin, β-catenin will not be sequestered and can translocate to
the nuclear to modulate gene expression, such as cyclin D1, c-myc and MMP-7, subsequently
activation of these genes leading to cell proliferation and invasion. As for desmosome
molecules, it has been reported that down regulation of desmosomal adhesion molecules,
desmoplakin and DSC1, correlates with invasion and metastasis without consideration of the
E-cadherin status, suggesting these molecules play a negative regulation role (Davies et al.,
1999; Natsugoe et al., 1997). However, the role of DSG3 in cellular function has not yet been
examined.
The role of another DSG3 interaction protein, plakoglobin has been recently studied in
regulation of cellular function, but the reports vary. First, several groups have reported that
plakoglobin acts as a tumor suppressor gene. This was initially inferred from the studies
showing loss of heterozygosity of the plakoglobin gene in certain types of tumors (Aberle et
al., 1995), reduced plakoglobin expression in various tumor types (Pantel et al., 1998;
Sommers et al., 1994; Syrigos et al., 1998) and over expression of plakoglobin can suppress
the tumorigenicity of mouse and human cells (Simcha et al., 1996). However, other reports
have different findings and suggested that plakoglobin possesses oncogenic properties. This
hypothesis originated from the observation that plakoglobin expression correlated positively
with the grade of hepatocellular carcinomas, being the highest in poorly differentiated tumors
(Endo et al., 2000). A positive correlation was also found between elevated expression of
plakoglobin and vascular invasion (Endo et al., 2000). Furthermore, the restoration of
plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and
tumorigenic potential (Rieger-Christ et al., 2005). It has been shown that plakoglobin also
reduce the expression desmocollin-2/3 and DSG2 leading to the suppression of cell migration
(Rieger-Christ et al., 2005). This suppression of cell motility by plakoglobin can be either
calcium-dependent or calcium-independent manner in keratinocytes (Yin et al., 2005)
Hypothesis of DSG3 signaling pathway in cell proliferation and invasion
In summary of the previous findings, two important points of conclusions can be drawn
that might associate with DSG3 signaling pathway. First, plakoglobin can suppression cell
migration and tumorigenicity and second, plakoglobin can translocation to nucleus to
participate Wnt signaling pathway. The armadillo proteins plakoglobin and plakophilins have
multiple functions inside and outside the desmosome. Plakoglobin is the nearest vertebrate
relative of β-catenin, which is a well-known downstream effector in the canonical Wnt
signaling pathway. Wnt signaling initiates a cascade of events that allow β-catenine to escape
the proteasome degradation machinery which normally ensures a low level of β-catenine
level in cytoplasm. β-Catenin can then translocate to the nucleus where it complexes with
LEF/TCF transcription factors and activates transcription of the target genes, such as c-myc,
cyclin D1, MMP-7 that that involved in development patterning, cell fate decisions, cell
proliferation, and survival (Figure 2A). Wnts are powerful regulators of cell proliferation and
differentiation, and their signaling pathway involves proteins that directly participate in both
gene transcription and cell adhesion. In this transduction pathway, β-catenin plays a central
role, which is a transcription cofactor with TCF/LEF (Figure 2B).
.
Since DSG3 is associated with plakoglobin in desmosome, over-expression of DSG3 in
cancer cells may lead to disrupting the balance of plakoglobin in cells. Therefore, I
hypothesize that in cancer cells, more plakoglobins may be captured by DSG3 in membrane
and less molecules in cytoplasm. This results to the reduction of the negative regulatory
effect of plakoglobin on TCF/LEF mediated transcription. Since plakoglobin plays an
antagonist role with β-catenin, the over-expression of DSG3 may lead to the elevation of
β-catenin in nuclear, which results to the increase of TCF/LEF regulate target gene
transcription and cell proliferation. I further hypothesize that the treatment of DSG3-RNAi
can reverse the downstream effect of DSG3. This results in plakoglobin translocate to the
nucleus, and render the negative regulatory effect of plakoglobin on TCF/LEF target gene
transcriptions (such as c-myc and cyclin D1,MMP-7), leading to the inhibition of cell
proliferation and cell migration, invasion. Model of this hypothesis is shown in the Figure 3.
Figure 2. Overview of the canonical Wnt-pathway and the possible interactions with
cadherin-mediated adhesion. (A) Secreted Wnt glycoproteins bind to Frizzled receptors and
LRP5 and 6 co-receptors to activate Dishevelled (Dsh) that subsequently acts to inhibit a
cytoplasmic complex involving GSK-3, Axin, β-catenin and APC. In the absence of Wnt
signals this complex mediates proteasomal degradation of β-catenin. Blocking of the
multiprotein complex by Dsh results in release and stabilization of β-catenin allowing its
nuclear transport and gene induction via binding to TCF/LEF transcription factors. (B) The
possible levels of interactions between Wnt signaling and cadherin-mediated adhesion.
(A)
(B)
Figure 3. Model of DSG3 signaling pathway that leading to up-regulation of cell proliferation. (A) In
normal cells, β-catenin and plakoglobin maintain a balanced ratio in nuclear to regulation TCF/LEF
turn on transcriptions. (B) In cancer cells, DSG3 is over-expressed which capture more molecules of
plakoglobins in membrane. Since lost of the antagonist function by plakoglobin in cytoplasm, more
molecules of β-catenin can enter nucleus, this subsequently turn on the TCF/LEF transcriptions. (C)
DSG3-RNAi treatment reverse the effects of DSG3, results in plakoglobin translocation to the
nucleus and rendering the negative regulatory effect of plakoglobin on TCF/LEF transcription.
Study aims and experimental designs
As state above, DSG3 has been hypothesized transduction of signals through the
desmosome interacting protein plakoglobin, which in turn compete β-catenin binding
on Tcf/Lef transcription factor (Figure 3), the following experiments will be designed
to investigate each step of the DSG3 signal pathway.
DSG3 knockdown disruptions the interaction between DSG3 and plakoglobin
DSG3 is a component of desmosome and will interacts with other desmosome
proteins to maintain intact extracellular and intraceullar cytoskeletons. Previously
reports show that through interaction with DSG3, plakoglobin contacts with
intermediate filaments to maintain cell-cell interaction (Figure 3C). Therefore, we
hypothesize that DSG3 signal is through plakoglobin pathway, and DSG3-siRNA will
disrupt DSG3 and plakoglobin interaction. For this, immunoprecipitation of DSG3
and immunoblotting of plakoglobin and the reverse interaction reactions will be
conducted. I will examine the interaction between DSG3 and plakoglobin and whether
the DSG3-RNAi treatment will decrease the interaction of these two molecules.
DSG3 knockdown increases plakoglobin translocation from cytoplasm to nuclear
In adherin junction, extracellular signal can stimulate intraceullar molecules
through plakoglobin signal after plakoglobin translocates to the nuclear to regulate
gene transcriptional activity. Therefore we hypothesize that when DSG3 is
over-expressed in cancer cells, most plakoglobin molecules will be tightly captured by
DSG3 at cell membrane, whereas plakoglobins can translocate to the nuclear when
DSG3 expression is knockdown by DSG3-siRNA. To examine this hypothesis,
immunofluorescent staining of the cellular section will be performed. In control
cancer cells, DSG3 and plakoglobin theoretically will be co-localized at cell
membrane. However, in the DSG3-RNAi treatment cells, the reduction of DSG3
expression will result to the increase of plakoglobin translocation into nuclear.
DSG3 knockdown increases the competitive binding of plakoglobin with
β-catenin to Tcf/Lef transcription factor
In Wnt signal pathway, the translocation of β-catenin to nuclear can increase
down stream Tcf/Lef transcriptional activity. Plakoglobin when translcoates to the
nuclear, theoretically, can compete binding with β-catenin to the Tcf/Lef binding site.
Therefore, we hypothesize that since most plakoglobins are tightly bound by DSG3 in
the DSG3 over-expressed cancer cells, most Tcf/Lef molecules are associated with
β-catenin. However, the binding of Tcf/Lef may be competed with plakoglobin when
DSG3 expression is knockdown by DSG3-siRNA. To examine this hypothesis,
immunoprecipitation with Tcf/Lef transcription factor and immunoblotting with either
plakoglobin or β-catenin will be performed. I propose that after increase plakoglobin
molecules in cells by DSG3 knockdown, the association of plakoglobin to Tcf/Lef
transcription factor will increase; whereas the association of β-catenin to Tcf/Lef will
decrease.
DSG3 knockdown reduces Tcf/Lef transcriptional activity
As stated above, plakoglobin when translcoates to the nuclear can compete
binding with β-catenin to the Tcf/Lef binding site leading to the reduction of the
transcriptional activity. Therefore, we hypothesize that since most plakoglobins are
tightly bound by DSG3 in the DSG3 over-expressed cancer cells, the suppression
signal of Tcf/Lef activity by plakoglobin is reduced. However, the suppression effect
of Tcf/Lef will be recovered when DSG3 expression is knockdown by DSG3-siRNA.
To examine this hypothesis, Tcf/Lef activity will be determined by luciferase reporter
assay. Co-transfection with the plasmids of Tcf/Lef luciferase reporter and either
DSG3-siRNA or control vector will be performed. I propose that after knockdown
DSG3 expression by siRNA, the Tcf/Lef transcriptional activity will reduce in
comparison with vector control.
DSG3 knockdown suppresses Tcf/Lef downstream target genes c-myc and cycline
D1, leading to the inhibition of cell proliferation
Tcf/Lef transcriptional factor regulates the expressions of many downstream
effectors including c-myc and cyclin D1 which control cell proliferation. Since DSG3
knockdown may reduce Tcf/Lef transcriptional activity, we hypothesize that the
expressions of Tcf/Lef downstream target genes of c-myc and cyclin D1 will be also
suppressed. This will lead to the inhibition of cell proliferation when DSG3 is
knockdown by siRNA. To examine this hypothesis, the expression levels of c-myc
and cyclin D1 will be determined after transfection of DSG3-siRNA plasmid. I
propose that after knockdown DSG3 expression by siRNA, the expressions of c-myc
and cyclin D1 will be reduced in comparison with vector control. Also, the cell
proliferation ability will be suppressed in DSG3-siRNA treated cells.
DSG3 knockdown suppresses Tcf/Lef downstream target gene MMP7, leading to
the inhibition of cell invasion
Tcf/Lef transcriptional factor regulates the expressions of many downstream
effectors including matrix metalloproteinase 7 (MMP7) which control cell invasion.
Since DSG3 knockdown may reduce Tcf/Lef transcriptional activity, we hypothesize
that the expressions of Tcf/Lef downstream target genes of MMP7 will be also
suppressed. This will lead to the inhibition of cell invasion when DSG3 is knockdown
by siRNA. To examine this hypothesis, the expression level of MMP7 and the
proteinase enzyme activity will be determined after transfection of DSG3-siRNA
plasmid. I propose that after knockdown DSG3 expression by siRNA, the expressions
of MMP7 as well as MMP enzyme activity will be reduced in comparison with vector
control. Also, the cell invasion ability will be suppressed in DSG3-siRNA treated
cells.
Results and discussion
As stated above, we hypothesize that DSG3 signal is through plakoglobin
pathway, and DSG3-siRNA will disrupt DSG3 and plakoglobin interaction. Using
immunofluorescent staining, we found that DSG3 and plakoglobin co-localized at cell
membrane in cancer cells (Figure 4). Immunoprecipitation and immunoblot results
further indicate that DSG3 and plakoglobin associate to each other in the cancer cell,
and DSG3 knockdown disrupt this interaction (Figure 5).
Our preliminary data also shown that DSG3 knockdown facilitates plakoglobin
translocate from cell membrane boundary to the nuclear (Figure 6). From luciferase
reporter assay, we further found that suppression of DSG3 expression can inhibit
Tcf/Lef transcriptional activity (Figure 7), and its down stream effect molecules, such
as cyclin D1 (Figure 8) and MMP7 (Figure 9) were also inhibited.
Our preliminary results are all in agreement and support our hypothesis that
desmosme complex protein DSG3 can mediate cell signal through plakoglobin.
Knockdown DSG3 expression by DSG3-siRNA can facilitate plakoglobin translocate
to the nuclear, and render a suppression effect on Tcf/Lef transcription activity,
leading to inhibition of cyclin D1 and MMP expression, which are involved in the
mechanism of cell proliferation and invasion.
Figure 4. DSG3 co-localized with plakoglobin. Immnuofluorescent staining with DSG3
(green) and plakoglobin (red) of OECM1 cells. Nuclear were stained with DAPI (blue). In the
merge figure can find DSG3 were co-localized with plakoglobin.
Figure 5. DSG3 knockdown disruptions the interaction between DSG3 and plakoglobin. (A) Cellular
extract proteins was immunoprecipitated (IP) with plakoglobin (Pg) and immunoblotted (IB) with
DSG3 to examine the potential interaction between these two molecules. Protein samples from
OEC-M1 cells (OEC-M1) or the cells transfected with vector (vector) shows high levels of DSG3
expression (lane 1) and strong association to each other (lane 2). However, protein samples from
DSG3-siRNA transfectecd cells (DSG3-siRNA) shows the reduction of DSG3 expression (lane 1) and
much weaker association between these two molecules (lane 2). (B) Control experiment showing that
DSG3-siRNA treatment has no effect on Plakoglobin expression (lane 1), as well as the successfulness
of immunoprecipitation experiment of plakoglobin (lane 2). N: negative control without protein
extraction in the IP experiment.
(A)
(B)
Figure 6. DSG3 knockdown increases plakoglobin translocation from cytoplasm to
nuclear. Cell transfect with vector control or DSG3-RNAi plasmid after 48 hrs then
fixed cell with formaldehyde and permeated with Triton-X100. Immunofluorescent
staining with plakoglobin. (A) Vector control (B) DSG3-RNAi treatment.
(A)
(B)
Figure 7. DSG3 knockdown reduces Tcf/Lef transcriptional activity. Cells were
contransfected with TCF/LEF luciferase reporter plasmid and RNAi vector or
DSG3-RNAi, and continuously culture for 24 or 48 hours. Cellular proteins were
extracted and subjected to luciferase activity assay using Steady-Glo luciferae reagent
(Promega).
Figure 8. DSG3 knockdown suppresses the expression of Tcf/Lef downstream target
genes c-myc and cycline D1. Cells were transfected with vector or DSG3-RNAi
plasmids and continuously cultured for 2 to 4 days. Cellular proteins were extracted
and subjected to immunoblot analysis of cyclin D1.
Figure 9. DSG3 knockdown suppresses the expression of Tcf/Lef downstream target
gene MMP7. Cells were transfected with vector or DSG3-RNAi plasmids and control
vector plasmid. Then reseed the equal cell numbers to the serum free medium and
collect medium to analysis MMP7 expression.