Download p53 expression in human colon cancer tumors in nude mice after

p53 expression in human colon cancer tumors in nude mice after siRNA CD44 gene
therapy
Shao Tiffany, Venkateswaran Subramaniam, Serge Jothy *
Department of Laboratory Medicine, St. Michael’s Hospital, Toronto, ON, Canada M5B 1W8
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON,
Canada
Abstract
Colorectal cancer is the second leading cause of cancer related deaths in North America.
CD44, a ubiquitously expressed transmembrane adhesion protein, is involved in fundamental
aspects of cancer cell biology such as tumor stem cell phenotype, cell adhesion and invasion, and
resistance to apoptosis. It is over-expressed in most human malignant tumors including human
colon cancer. Another protein with a major implication in colorectal carcinogenesis is p53, a
tumor suppressor protein. It is widely known to play an important role in the control of cell cycle
and apoptosis and is often referred to as a “guardian of the genome”. Mutations in the p53 gene
result in decreased genetic stability, reduced apoptosis, and increased survival and propagation of
cells with damaged DNA. The nuclear accumulation of the p53 protein has been reported in a
variety of malignant tumors, including colon cancer. The number of mutations in p53 was found
to increase as colorectal adenomas progress to carcinomas to metastatic carcinoma. To further
delineate the role of p53 and CD44 in colon cancer, immunohistochemistry studies were carried
out using tumors generated by HT-29 human colon cancer cells in nude mice after siRNA CD44
gene therapy, as compared to untreated control xenografts. Using this xenograft mouse model
with reduced CD44 expression after siRNA CD44 gene therapy, we were able to demonstrate a
significant decrease in p53 expression that is associated with the inhibition of CD44 expression
in tumors derived from human colon cancer cells. This study suggests that the association
between decreased CD44 expression and decreased p53 expression could be a possible
manifestation of a return to a less aggressive tumor phenotype after siRNA CD44 gene therapy,
providing insights into the likely roles of CD44 and p53 in the regulation of apoptosis and cancer
progression.
Keywords: siRNA; CD44; p53; Colon cancer; Gene therapy; xenograft model; HT-29 cells
(Subramaniam et al., 2005). Epithelial cell
Introduction
turn over in the colon involves a 6-day
Colorectal cancer is the second leading
migration of the cells generated by stem
cause of cancer related mortality in North
cells from the base of the crypt to cell
America. Transformation of the normal
detachment, and apoptosis at the surface
colonic epithelium to an adenoma and then
epithelium.
to a carcinoma is the result of a sequence of
multiple
steps.
is
CD44, a ubiquitously expressed cell
of
adhesion molecule, facilitates both cell-cell
molecular alterations, including alterations
and cell-matrix interactions in normal and
in epithelial cell differentiation, proliferation,
transformed epithelial cells (Ponta et al.,
and apoptosis. The latter two processes are
2003; Rudzki et al., 1997). Dysregulations
highly regulated in the constantly renewing
in cell adhesion to neighbouring cells and
untransformed
The
extracellular matrix can result in apoptosis
maintenance of homeostasis of the colonic
(Frisch et al., 1994; Grossman et al., 2002;
epithelium involves adhesion molecules and
Shanmugathasan and
regulators of cell cycle and apoptosis
normal
characterized
by
This
a
colonic
sequence
large
number
epithelium.
colonic
Jothy, 2000).
epithelium,
CD44
In
is
expressed in dividing cells at the base of the
RNAi. Intratumoral RNAi of CD44 has been
crypt but not the nonproliferating epithelium
shown to suppress tumor growth and
of the upper crypt and luminal surface
increased
(Lakshman et al., 2005). It is frequently
(Subramanian et al., 2007). Based on this
overexpressed at both the mRNA and
previous mouse xenograft model, siRNA
protein level in colon cancer and other types
targeting of a discrete sequence of human
of carcinomas (Fox et al., 1994; Wielenga et
CD44 provides a potential therapeutic
al., 1993, 2000; Woodman et al., 1996).
approach for colon cancer.
apoptosis
in
nude
mice
CD44 is also found to be associated with the
characteristic tumor stem cell phenotype and
One of the most common genetic changes
the modulation of the directional motility
in colorectal cancer involves the p53 tumour
and migration of human colon cancer cells.
suppressor gene (Greenblatt et al., 1994).
(Jin et al., 2006; Lakshman et al., 2004a,b,
The p53 gene is located on the short arm of
2005; Shipitsin et al., 2007; Wong et al.,
chromosome 17, and its protein product acts
2003). Recently, it has been suggested that
as a transcription factor to maintain genetic
CD44 provides resistance to apoptosis in
integrity. It does so by arresting cell cycle at
human colon tumors xenotrasplanted in mice
the G1-S transition for DNA repair and
after siRNA mediated CD44 knock down
induction of apoptosis, resulting in tumor
(Subramaniam et al., 2007).
growth suppression. (Kastan et al., 1991).
Conversely, allelic loss and mutations in the
Previous
studies
in
our
laboratory
p53 gene result in decreased genetic stability,
employed the strategy of inhibiting CD44
reduced apoptosis, and increased survival
mRNA expression using a method based on
and propagation of cells with damaged DNA
(Soong wt al., 1997). The number of
this
mutations in p53 thus increases as colorectal
demonstrated that a significant decrease in
adenomas
to
p53 expression is associated with the
metastatic carcinoma (Goh et al., 1994;
inhibition of CD44 expression in tumors
Scott et al., 1995). The wild type p53 protein
derived from human colon cancer cells.
progress
to
carcinomas
mouse
xenograft
model,
we
has a short half-life and is removed rapidly
from the nucleus, while the mutant p53 has a
prolonged half-life and accumulates in the
nucleus,
where
it
is
detectable
Materials and Methods
siRNA CD44 gene therapy tumor samples
by
immunohistochemistry (Leahy et al., 1996).
The nuclear accumulation of the p53 protein
has been reported in a variety of malignant
tumors, including colon cancer (Bartek et al.,
1991). Furthermore, a relation between p53
overexpression and poor prognosis has been
shown in many types of human cancers,
Human colon cancer tumors derived from
the HT29 colon cancer cell line, were
xenotransplanted into nude mice and treated
by siRNA CD44 gene therapy were obtained
according to the published protocol of
Subramaniam,V., et al., 2007.
Antibodies
such as colon cancer (Manne et al., 1997).
Rabbit anti-human p53 antibody and
rabbit IgG control serum (Santa Cruz
In the present study, we performed
Biotechnology, Santa Cruz, CA) were used
immunohistochemical experiments to further
for immunohistochemistry experiments at a
investigate p53 expression of human colon
concentration of 4 µg/mL. The secondary
cancer tumors xenotransplanted in nude
antibody used was Polymer-HRP labelled
mice after siRNA CD44 gene therapy. Using
goat anti-rabbit IgG (Dako, Carpinteria, CA).
Immunohistochemistry
diaminobenzidine (DAB) substrate (Dako,
Carpinteria, CA) as chromogen. Sections
Paraffin embedded sections of human
were then dehydrated and mounted with
colon cancer tumors in nude mice (either
Permount (Fisher Scientific, Ottawa, ON).
after mock transfection or after siRNA
As
CD44 gene therapy) were dewaxed and
immunostaining, a human breast cancer
rehydrated. Antigen retrieval was performed
tissue was used. Negative controls for all
with sodium citrate buffer (pH6.0) in a
immunostaining
pressure cooker for 30 min followed by
substituting the primary antibody with rabbit
cooling for 20 min at room temperature.
IgG
Endogenous peroxidase was blocked with
concentration. Each slide was first scanned
1% H2O2 in methanol for 10 min at room
with a low magnification, and 3 tumor fields
temperature. Sections were incubated in 5%
were
normal goat serum to block non-specific
measurements at 20X power magnification.
binding sites, followed by incubation with
Quantification of the p53 immunostaining
primary p53 antibody for 1 hr at room
was performed by calculating the percentage
temperature and then overnight at 4°C. After
of positive nuclei of tumour cells in relation
washing with 1X PBS, sections were
to the area of the representative fields (40
incubated with secondary antibody for 1 hr
000 um2). Statistical analysis was carried out
at room temperature. Immunoperoxidase
using the Student’s t-test.
detection
was
achieved
using
a
positive
at
the
selected
control
were
same
for
carried
for
out
p53
by
immunoglobulin
observation
and
Results
Reduction of p53 expression after siRNA
nuclearimmunostaining
for
p53
was
CD44 gene therapy of human colon cancer
significantly reduced by 16% in the tumors
tumors in nude mice
that had received the siRNA CD44 gene
therapy, when compared to the mock
We measured the expression of p53 in our
xenograft mouse model and found that
A
transfected vector control group (Figure 1,
Figure 2, p<0.05).
B
Fig.1. p53 expression is reduced in human colon cancer tumors in nude mice after siRNA
CD44 gene therapy: Immunohistochemical staining showed a reduction of p53 expression in
siRNA CD44 treatment group (A), when compared to the mock transfected vector control group
(B). Positive dark brown staining indicates p53 expression (p53 immunostaining, no
counterstaining, x20 magnification).
Fig.2. Human colon cancer tumors in nude mice after siRNA CD44 gene therapy shows
decreased p53 expression: A significant (*p<0.05) reduction in the number of p53 positive
nuclei occurs after siRNA CD44 knock down, when compared to the vector control. Data
represents the average of 4 independent experiments.
plays an anti-apoptotic role as an oncogenic
Discussion
It is well documented that apoptosis is
defective
in
colonic
adenomas
and
carcinomas, and it can be induced by
disrupting
cellular
interaction
with
protein in cancer metastasis (Wielenga et al.,
1993; Mulder et al., 1997; Lakshman et al.,
2004a,b, 2005 ).
In
our
previous
study,
we
neighbouring cells and the matrix of
demonstrated that the expression of CD44
basement membranes (Bedi et al., 1995;
can be significantly suppressed in HT-29
Partik et al., 1998). Thus, upregulation of
human colon cancer cells by siRNA CD44
CD44 expression, a cell adhesion protein,
inhibition in a xenograft mouse model
facilitates these cellular interactions and
(Subramaniam et al, 2007). HT-29 cells
express one of the highest levels of CD44
suggest that the main mechanism of the
among human colon cancer cell lines. The
therapeutic effect of CD44 siRNA inhibition
CD44 sequence susceptible for siRNA
is through increased apoptosis of tumour
inhibition is located at the junction of
cells by reversing the anti-apoptotic effect of
transcripts encoded by standard exons 16
CD44, involving caspase 3 and poly (ADP-
and 17 (Screaton et al., 1992). In this study,
ribose)
intratumoral delivery of siRNA CD44 was
caspase 3 is found to mediate the cleavage
accomplished using polyethyleneimine (PEI).
of PARP and apoptosis in human colon
PEI is a cationic, nontoxic polymer, which
cancer cells. In our previous study, we
retains its physical characteristics without
observed a significant increase in levels of
eliciting a significant immune response
active caspase 3 and PARP in the tumors
(Guan et al., 2005). The injection of
after siRNA CD44 gene therapy when
PEI/siRNA CD44 resulted in significant
compared to vector control. Taken together,
reduction in tumor growth compared with
the data above and the anti-apoptotic effect
tumor
with
of CD44 suggest that the tumor growth
PEI/siRNA vector. The siRNA CD44 clones
suppression after siRNA CD44 inhibition
were less adhesive than vector control,
might be due to the promotion of tumor cell
suggesting a loss of function for CD44.
apoptosis (Subramaniam et al., 2007).
bearing
mice
injected
polymersase
(PARP).
Cleaved
These xenografted tumors after siRNA
CD44 gene therapy had reduced level of
CD44, decreased tumor mass and tumor
growth, and increased tumor apoptosis.
These results from this xenograft model
Due to the fundamental role of p53 in
cancer cell biology, it has been studied by
different methods such as xenografts and
tumor cell lines and tissues in a variety of
human malignancies (Kaklamanis et al.,
The significant correlation between the
1993). Abnormalities in p53 expression have
overexpression of p53 protein and the
been studied in particular detail in colorectal
histologic grade, tumor size, serosal invasion,
cancer. The normal p53 protein has a short
lymphatic invasion, venous invasion, lymph
intracellular half-life of 6-20 minutes,
node
whereas many missense mutations found in
remains controversial (Chang et al., 1996).
cancer cells induce conformational changes
Numerous studies have investigated the
resulting in a mutant p53 protein with a
significance of p53 expression and its
longer half-life of up to 6 h (Clarke et al.,
relation to CD44 expression in human
1988; Dermot et al., 1996; Rogel et al., 1985;
cancer, providing contradictory results on
Sturzbecher et al., 1987). This functionally
the conflicting expressions between CD44
inactive and stabilized p53 protein can be
and p53 (Darai et al., 1998; Formby et al.,
detected
by
1999; Kuncova et al., 2007; Endo et al.,
immunohistochemical methods (Roviello et
2000; Kim et al., 1994; Mulder et al., 1995;
al., 1999).
Zavrides et al., 2005). For example, it has
in
the
cell
nucleus
metastasis,
or
colon
carcinomas
been shown that there is a correlation of
In previous immunohistochemistry studies,
CD44v6 expression with a higher incidence
there have been conflicting results on the
of p53 expression in various forms of human
prognostic
cancers, including colon cancer (Darai et al.,
significance
of
p53
overexpression and its association with poor
1998;
Kanenori
et
al.,
2000).
CD44
patient survival in colorectal cancer (Leahy
expression in colorectal adenomas is an
et al., 1996; Diez et al., 2000; Soong et al,
early event prior to p53 gene mutation and it
1997; Ofner et al., 1995; Wolf et al., 1997).
has been suggested that aberrant p53
expression
may
on
p53 expression of human colon cancer
upregulation of CD44v6 isoform (Kim et al.,
tumors in nude mice after siRNA CD44
1994). However, other studies have provided
gene therapy. We exploited our previous
contradicting results with respect to the
observation
correlation
p53
expression by siRNA CD44 gene therapy,
expression (Formby et al., 1999; Jitka et al.,
results in increased apoptosis (Subramaniam
2007). These studies have raised the
et al., 2007). Using this mouse xenograft
questions of the true frequency of p53
model, we demonstrate in this study that a
expression, its relationship to CD44, what
significant decrease in the frequency of p53
association it has with concurrent colon
expression is associated with a reduction of
carcinomas, and its mechanistic role in the
CD44 after siRNA CD44 gene therapy,
context of apoptosis in colon cancer.
consistent with the therapeutic pressure that
However, few studies have analyzed the
was used. A possible explanation for the
correlation of p53 and CD44 expression in
observed decrease in p53 expression in
the context of apoptosis or looked into the
xenografted human tumors could be that it is
possible role of p53 in relation to CD44 in
caused by decreased signalling from CD44.
the mechanistic regulation of apoptosis.
Globally, p53 expression in human colon
between
be
dependent
CD44
and
that
reduction
of
CD44
cancer cells might be dependent on CD44, in
The present study has undertaken to
addition to other cellular signals altering p53.
examine one of these questions: the specific
Furthermore, in the case of a complete
role of p53 in relation to CD44 in the
reversion of the tumor cell phenotype to a
context of apoptosis. Immunohistochemistry
normal cell phenotype, there would be close
experiments were performed to investigate
to no CD44 expression or p53 expression.
Therefore,
the
association
between
decreased CD44 expression and decreased
p53 expression could be a manifestation of a
return to a less aggressive tumor phenotype
due to the therapeutic effect of siRNA CD44
gene therapy. Overall, this study was the
first to look at p53 expression after siRNA
CD44 gene therapy in xenografted human
tumors in nude mice and it provides insights
into the possible roles of CD44 and p53 in
the regulation of apoptosis. However, more
studies are necessary to further delineate the
possible linking of p53 and CD44 in the
regulation of apoptotic process in colorectal
cancer.
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Acknowledgement
I
thank
Dr.
Venkateswaran
Sergy
Jothy
Subramaniam
guidance and support.
Conflict of Interest
There was no conflict of interest.
and
for
Dr.
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