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Cancer Patent Abstract
The present invention provides an agent for inhibiting metastasis
of colorectal cancer and a method for inhibiting metastasis of colorectal
cancer, which inhibit the function of Asef (i.e., binding activity
to the APC gene product or guanine nucleotide exchange factor activity)
that binds to the gene product of the tumor suppressor gene APC
that plays an important role in tumorigenesis and in developmental
processes, and/or inhibit the expression of the Asef gene.
Cancer Patent Claims
What is claimed is:
1. An oligonucleotide consisting of the nucleotide sequence set
forth in SEQ ID NO: 1 in the sequence listing.
Cancer Patent Description
This application is a National Stage Application of PCT/JP2003/010449,
filed Aug. 19, 2003 and claims priority from Japanese Patent Application
No. 2002-382083, filed Nov. 24, 2002, which is incorporated herein
by reference in its entirety.
TECHNICAL FIELD
The present invention relates to a method for inhibiting metastasis
of colorectal cancer and an agent for inhibiting metastasis of colorectal
cancer, which are characterized by inhibiting the function of Asef
(APC-stimulated guanine nucleotide exchange factor) and/or inhibiting
the expression of Asef. More specifically, the present invention
relates to an agent for inhibiting metastasis of colorectal cancer,
an agent for inhibiting Asef, a pharmaceutical composition, an agent
for preventing and/or treating colorectal cancer, a method for inhibiting
metastasis of colorectal cancer, and a method for preventing and/or
treating colorectal cancer, which are characterized by inhibiting
the expression of Asef, inhibiting the binding of Asef to the gene
product of APC (Adenomatous Polyposis Coli), or inhibiting the guanine
nucleotide exchange factor (hereunder, referred to in abbreviated
form as "GEF") activity of Asef.
BACKGROUND ART
Asef is a protein that was found by the present inventors as a
colorectal tumor suppressor gene-associated protein M1, which has
already been disclosed and for which a patent application has been
filed (Patent Reference 1 and Non-patent Reference 1). The protein
consists of 619 amino acid residues, and contains the Db1 homology
(DH) domain, the pleckstrin homology (PH) domain and the Src homology
3 (SH3) domain in its amino acid sequence.
In terms of function, it is known that Asef has GEF activity specific
for Rac. Rac belongs to the Rho family, which is one of the small
GTP-binding protein families. More specifically, Asef binds to Rac
to stimulate a GDP/GTP exchange reaction which results in the activation
of Rac, thereby acting on NF.kappa.B, c-jun, SRE and the like, which
are located downstream of the Rac related-intracellular signal transduction.
The Rho family proteins play key roles in the reorganization of
the acting network, thereby regulating cell migration and cell-cell
adhesion. Therefore, there is a possibility that Asef induces cellular
lamellipodia (lobopodium) or cell membrane ruffling and participates
in cell migration and cell-cell adhesion.
It has been revealed that the binding of Asef to the gene product
of the tumor suppressor gene APC via the armadillo repeat domain
of the gene product. The GEF activity of Asef is positively regulated
by the APC gene product. Actually, the induction of Asef-mediated
cell membrane ruffling or lamellipodia formation by the APC gene
product is observed in MDCK cells that are canine kidney-derived
epithelial-like cells. Further, Asef accumulates at the tips of
microtubules in motile cells similarly to the APC gene product.
Therefore, Asef may hold the key to control cell migration when
cells migrate from the crypt to the villus tip of the colon.
Meanwhile, the tumor suppressor gene APC (Non-patent Reference
2) has been isolated as a responsible gene for familial adenomatous
polyposis (FAP). Mutation of the gene is observed in approximately
70% to 80% of sporadic colorectal cancers. The APC gene product
(hereunder, referred to as "APC") is a giant protein of
approximately 300 kDa that comprises 2,843 amino acid residues.
APC contains an armadillo repeat domain in the amino acid sequence
thereof that participates in protein-protein interaction. Most somatic
APC mutations observed in colorectal tumor cells occur within its
central region called the "mutation cluster region (MCR)"
and result in the generation of truncated APCs that lack the binding
sites for microtubules, EB 1 or hDLG, and at least some of the sites
for .beta.-catenin and Axin (Non-patent References 4, 5, 6, 7 and
8). However, the region of APC responsible for binding to Asef,
the armadillo repeat domain, is retained in most mutant APCs (Non-patent
References 6, 7 and 8). APC has a function to bind to .beta.-catenin,
one kind of oncogene product, to induce its degradation (Non-patent
References 2, 3, 4, 5 and 6). .beta.-catenin, which is a Wnt/Wingless
signal transduction factor, binds to the cytoplasmic domain of cadherin
and plays a role in cell adhesion, while it plays important roles
in developmental processes and in tumorigenesis (Non-patent References
9 and 10).
The amino acid sequence of Asef and the nucleotide sequence of
its gene have been deposited with GenBank under the accession number
AB042199. Further, the amino acid sequence of APC and the nucleotide
sequence of its gene have been deposited with GenBank under the
accession number NM000038.
Documents referred to in this specification are listed hereunder:
Patent Reference 1: Japanese Patent Laid-Open No. 2001-057888. Non-patent
Reference 1: Kawasaki, Y., et al., Science, 2000, Vol. 289, p. 1194-1197.
Non-patent Reference 2: Kinzler, K. W., et al., Cell, 1996, Vol.
87, p. 159-170. Non-patent Reference 3: Fearnhead, et al., Human
Molecular Genetics, 2001, Vol. 10, p. 721-733. Non-patent Reference
4: Bienz, M., et al., Cell, 2000, Vol. 103, p. 311-320. Non-patent
Reference 5: Perifer, M., et al., Science, 2000, Vol. 287, p. 1606-1609.
Non-patent Reference 6: Akiyama, T., Cytokine and Growth Factor
Reviews, 2000, Vol. 11, p. 273-282. Non-patent Reference 7: Miyoshi,
Y., et al., Human Molecular Genetics, 1992, Vol. 1, p. 229-233.
Non-patent Reference 8: Nagawa, H., et al., Human Mutation, 1993,
Vol. 2, p. 425-434. Non-patent Reference 9: Cell, 1996, Vol. 86,
p. 391-399. Non-patent Reference 10: Nature, 1996, Vol. 382, p.
638-642. Non-patent Reference 11: Wong, M. H., et al., Proceeding
of national academy of science USA.right brkt-bot. 1996, Vol. 93,
p. 9588-9593. Non-patent Reference 12: Oshima, H., et al., Cancer
Research, 1997, Vol. 57, p. 1644-1649. Non-patent Reference 13:
Paddison, P. J., et al., Genes and Development, 2002, Vol. 16, p.
948-958.
DISCLOSURE OF THE INVENTION
It is known that Asef binds to the gene product of the tumor suppressor
gene APC which plays important roles in tumorigenesis and in developmental
processes as described in the foregoing. However, the function of
Asef in cells and the relation of Asef with diseases have not yet
been clarified. To clarify the function of Asef and regulate the
function thereof makes it possible to prevent and treat diseases
attributable to Asef.
The present inventors hypothesized based on the GEF activity of
Asef and its intracellular localization that Asef may participate
in cell migration and cell-cell adhesion, and found that Asef promotes
the motility of colorectal tumor cells in colorectal cancers, particularly
in colorectal cancers in which APC mutations are observed, and participates
in the metastasis. By utilizing this finding, the present inventors
found that metastasis of colorectal cancer is inhibited by inhibiting
the function of Asef and/or inhibiting the expression of the Asef
gene, and thereby complete the present invention.
That is, one aspect of the present invention relates to an agent
for inhibiting metastasis of colorectal cancer, wherein the agent
inhibits the function of Asef and/or inhibits the expression of
the Asef gene.
Another aspect of the present invention relates to an agent for
inhibiting metastasis of colorectal cancer, wherein the agent inhibits
the expression of the Asef gene.
A further aspect of the present invention relates to an agent for
inhibiting metastasis of colorectal cancer, wherein the agent inhibits
the binding of Asef to the gene product of APC.
A still further aspect of the present invention relates to an agent
for inhibiting metastasis of colorectal cancer, wherein the agent
inhibits the guanine nucleotide exchange factor activity of Asef.
A further aspect of the present invention relates to a method for
inhibiting metastasis of colorectal cancer, wherein the method comprises
inhibiting the function of Asef and/or inhibits the expression of
the Asef gene.
A further aspect of the present invention relates to a method for
inhibiting metastasis of colorectal cancer, wherein the method comprises
inhibiting the expression of the Asef gene.
A still further aspect of the present invention relates to a method
for inhibiting metastasis of colorectal cancer, wherein the method
comprises inhibiting the binding of Asef to the gene product of
APC.
A further aspect of the present invention relates to a method for
inhibiting metastasis of colorectal cancer, wherein the method comprises
inhibiting the guanine nucleotide exchange factor activity of Asef.
A further aspect of the present invention relates to an agent for
inhibiting Asef, wherein the agent utilizes RNA interference for
the expression of the Asef gene.
A still further aspect of the present invention relates to an agent
for inhibiting Asef, comprising an oligonucleotide that exhibits
an RNA interference effect on the expression of the Asef gene.
A further aspect of the present invention relates to an oligonucleotide
having the nucleotide sequence set forth in SEQ ID NO: 1 in the
sequence listing.
A further aspect of the present invention relates to an oligonucleotide
having the nucleotide sequence set forth in SEQ ID NO: 2 in the
sequence listing.
A still further aspect of the present invention relates to an oligonucleotide
having the nucleotide sequence set forth in SEQ ID NO: 3 in the
sequence listing.
A further aspect of the present invention relates to an oligonucleotide
having the nucleotide sequence set forth in SEQ ID NO: 4 in the
sequence listing.
A further aspect of the present invention relates to the preceding
agent for inhibiting Asef, comprising an oligonucleotide having
the nucleotide sequence set forth in SEQ ID NO: 1 or 3 in the sequence
listing.
A still further aspect of the present invention relates to a method
for inhibiting Asef, wherein the method utilizes RNA interference
on the expression of the Asef gene.
A further aspect of the present invention relates to a method for
inhibiting Asef, wherein the method comprises utilizing an oligonucleotide
exhibiting an RNA interference effect on the expression of the Asef
gene.
A further aspect of the present invention relates to the preceding
method for inhibiting Asef, wherein the method comprises utilizing
an oligonucleotide having the nucleotide sequence set forth in SEQ
ID NO: 1 or 3 in the sequence listing.
A still further aspect of the present invention relates to an agent
for inhibiting metastasis of colorectal cancer, comprising any one
of the preceding agents for inhibiting Asef.
A further aspect of the present invention relates to an agent for
inhibiting metastasis of colorectal cancer, comprising an oligonucleotide
having the nucleotide sequence set forth in any one of SEQ ID NOS:
1 to 4 in the sequence listing.
A further aspect of the present invention relates to a method for
inhibiting metastasis of colorectal cancer, wherein the method uses
any one of the preceding agents for inhibiting Asef.
A still further aspect of the present invention relates to a method
for inhibiting metastasis of colorectal cancer, wherein the method
uses an oligonucleotide having the nucleotide sequence set forth
in any one of SEQ ID NOS: 1 to 4 in the sequence listing.
A further aspect of the present invention relates to a pharmaceutical
composition, comprising any one of the preceding agents for inhibiting
metastasis of colorectal cancer, or any one of the agents for inhibiting
Asef.
A further aspect of the present invention relates to an agent for
preventing and/or treating colorectal cancer, comprising any one
of the preceding agents for inhibiting metastasis of colorectal
cancer, or any one of the agents for inhibiting Asef.
A still further aspect of the present invention relates to a method
for preventing and/or treating colorectal cancer, wherein the method
uses any one of the preceding agents for inhibiting metastasis of
colorectal cancer, or any one of the agents for inhibiting Asef.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates decreased cell-cell adhesion of MDCK cells infected
with adenoviruses that contains DNA encoding Asef. As shown in the
vertical axis, cell-cell adhesion is represented by a numerical
value obtained by dividing the number of cell clumps (Np) by the
total number of cells (Nc). Cells were infected with adenoviruses
containing a gene encoding full-length Asef (denoted by "Asef-full"),
a gene encoding the armadillo repeat domain of the APC (denoted
by "APC-arm"), a gene encoding an Asef mutant that lacks
the APC-binding region (denoted by "Asef-.DELTA.APC"),
or a gene encoding an Asef mutant that lacks the DH domain (denoted
by "Asef-.DELTA.DH"), as shown in the figure. The results
are shown as mean.+-.standard deviation (SD) obtained over three
independent experiments.
FIG. 2 illustrates that enhanced MDCK cell motility by the expression
of the Asef gene was further increased by co-expression with an
APC mutant that contains the armadillo repeat domain (APC-arm, APC-876
and APC-1309), while the enhanced motility by expression of the
Asef gene, as well as the inherent cell motility, was decreased
by expression of the Asef-.DELTA.DH gene or the Asef-ABR (APC-binding
region of Asef) gene. The results are shown as the relative migration
as compared to that of the parental cells. The term "Mock"
in the figure refers to cells transfected with an empty vector.
FIG. 3a illustrates the binding of Asef to truncated APC mutants
in SW480 cells. Analysis of binding was carried out by immunoprecipitation
using an anti-Asef antibody (Anti-Asef). In the figure, the symbol
+indicates that the antibody used was pre-incubated with antigen
before immunoprecipitation.
FIG. 3b illustrates that Asef-ABR (APC-binding region of Asef)
inhibited the interaction of IVT-APC-arm with GST-Asef-full in vitro
in a dose dependent manner. In the figure, "MW." represents
a molecular marker.
FIG. 4 illustrates that the motility of colorectal tumor SW480
cells is enhanced by expression of the Asef gene or the gene encoding
Asef that lacks the APC-binding region (Asef-.DELTA.APC), while
the motility of various colorectal tumor cells (SW480, DLD-1, HCT15,
WiDr and HCT116) did not change or decreased when the gene encoding
Asef lacking the GEF domain (Asef-.DELTA.DH) was expressed. The
results are shown as the relative migration as compared to that
of each cell expressing the LacZ gene as a control.
FIG. 5 illustrates that the short hairpin RNAs, shRNA-Asef and
shRNA-APC which inhibit the expression of the Asef gene and the
APC gene respectively, both decreased the motility of colorectal
tumor cells having an APC mutation (SW480 and WiDr), while no effect
was observed on the motility of colorectal tumor cells having normal
APC (HCT116 and LS180). As controls for comparison, the short hairpin
RNAs, mut-shRNA-Asef and mut-shRNA-APC, which do not inhibit the
expression of Asef gene or APC gene, were used. The results are
shown as the relative migration as compared to that of each cell
transfected with mut-shRNA-Asef.
DETAILED DESCRIPTION OF THE INVENTION
The present invention claims the benefit of priority from Japanese
Patent Application No. 2002-382083, which is incorporated herein
by reference in its entirety.
Technical and scientific terms used herein have the meanings as
normally understood by those skilled in the art, unless otherwise
defined. Various methods that are well known to those skilled in
the art are referenced herein. These reference materials, such as
published materials disclosing known methods cited herein, are incorporated
herein by reference in their entirety.
Embodiments of the present invention are explained in further detail
below. However, the detailed description below is exemplary and
for the purpose of explanation only, and is not intended to limit
the scope of the present invention.
In the present invention, it was found that Asef decreases cell-cell
adhesion of epithelium-derived cells and also noticeably promotes
the motility thereof. Further, it was found that these functions
are regulated by APC, and particularly, it was found that truncated
APC mutants that are identified in the majority of colorectal tumor
cells activate Asef constitutively. Therefore, it is believed that
formation of a complex between mutated APC and Asef contributes
to aberrant motility of colorectal tumor cells. That is, it is concluded
that the complex may be involved in the upward migration of intestinal
epithelial cells, more specifically, in the migration from the crypt
to the villus tip. Indeed, it has been reported that forced expression
of the APC gene induces aberrant cell migration in the intestinal
epithelium (Non-patent Reference 11). It has been reported that
early adenoma cells in APC knockout mice exhibit a proliferation
rate similar to that of normal crypt epithelial cells, but lack
directed migration along the crypt-villus axis (Non-patent Reference
12).
Aberrant migratory behavior due to Asef activation by truncated
APC mutants may be thus significant for both adenoma formation and
tumor progression to invasive malignancy. In addition, Asef mutants
that lack the GEF domain do not decrease cell-cell adhesion or do
not promote cell motility, resulting in the conclusion that GEF
activity is important for such a function of Asef.
In the present invention, it was revealed that the motility of
colorectal tumor cells expressing mutant APCs can be inhibited by
using a dominant-negative mutant that inhibits the binding of Asef
to mutant APCs, for example, a mutant consisting of the APC-binding
region (amino acid sequence from the 73.sup.rd to the 126.sup.th
amino acid residue) in the amino acid sequence of Asef or a mutant
that lacks the GEF domain of Asef. Also revealed was that the motility
of colorectal tumor cells expressing mutant APCs can similarly be
inhibited by inhibiting the expression of the Asef gene or the APC
gene. Further, it was found in an in vivo study using severe combined
immunodeficient mice (SCID mice) that the tumorigenicity, proliferative
growth and, moreover, metastasis of human colorectal tumor cells
expressing the aforementioned dominant-negative mutants are inhibited
in comparison to those of the cells that do not express the mutants.
Such an inhibition was observed similarly in a study using cells
that were obtained by cloning after expression of the mutants as
human colorectal tumor cells expressing the dominant-negative mutants,
and also in a study (mixed-population method) using a mixed population
that was obtained from cells transformed with the labeled mutants
by using a cell sorter to concentrate the cells expressing the mutants
to a density of 90% or more employing the label as an indicator.
The inhibition of the function of Asef thus makes it possible to
inhibit the motility of cells and, further, to inhibit tumorigenicity
of cells and proliferative growth and/or metastasis of tumor cells.
Since the inhibition of cell motility can also be achieved by inhibiting
the expression of the Asef gene or the APC gene, it is possible
to inhibit tumorigenicity of cells and proliferative growth and/or
metastasis of tumor cells by inhibiting the expression of each these
genes.
Based on the above-described findings, the present invention provides
an agent for inhibiting metastasis of colorectal cancer and a method
for inhibiting metastasis of colorectal cancer, which are characterized
by inhibiting the function of Asef. The agent for inhibiting metastasis
of colorectal cancer and the method for inhibiting metastasis of
colorectal cancer are characterized by inhibiting the function of
Asef and/or inhibiting the expression of the Asef gene.
Inhibition of the expression of the Asef gene can be carried out,
for example, by applying an RNA interference effect on the expression
of the Asef gene. RNA interference is a method for inhibiting the
expression of a gene by using RNA, as has been reported in recent
years (Non-patent Reference 13). More specifically, the expression
of the Asef gene can be inhibited by using an oligonucleotide that
exhibits an RNA interference effect on the expression of the Asef
gene. Examples of the oligonucleotide can include a cDNA having
the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence
listing. The complementary RNA (SEQ ID NO: 3 in the sequence listing)
of the cDNA can also be used. Inhibition of the expression of the
Asef gene can be carried out by transfecting a cell with a vector
containing the cDNA or with the complementary RNA thereof. Transfection
of a cell with the vector or with the RNA can be conducted utilizing
a known method such as lipofection. Accordingly, an agent for inhibiting
Asef comprising the aforementioned oligonucleotide is also included
in the scope of the present invention. The agent for inhibiting
Asef may contain one kind of oligonucleotide, or may contain two
or more kinds of oligonucleotide. Further, inhibition of the Asef
gene expression may also be carried out by using an antisense oligonucleotide
against the Asef gene. The aforementioned oligonucleotide exhibiting
an RNA interference effect or the aforementioned antisense oligonucleotide
can be obtained from oligonucleotides that are designed on the basis
of the nucleotide sequence of the Asef gene, by selecting oligonucleotides
that specifically inhibit the expression of Asef using an Asef gene
expression system.
Inhibition of the function of Asef can be carried out, for example,
by inhibiting the binding of Asef to APC, or inhibiting the GEF
activity of Asef. The binding of Asef to APC, which is the target
of inhibition, is preferably the binding of Asef to normal APC,
more preferably the binding of Asef to an APC mutant, further preferably
the binding of Asef to a truncated APC mutant, and still more preferably
the binding of Asef to a truncated APC mutant that contains an armadillo
repeat domain. Examples of a truncated APC mutant that contains
an armadillo repeat domain include a polypeptide consisting of the
consecutive amino acid residues from the 1.sup.st (the N terminus)
to the 876.sup.th residue of the amino acid sequence of APC, or
a polypeptide consisting of the consecutive amino acid residues
from the 1.sup.st (the N terminus) to the 1309.sup.th residue of
the amino acid sequence of APC. These polypeptides were identified
as truncated APC mutants in most colorectal cancers and familial
adenomatous polyposis (FAP).
Inhibition of the binding of Asef to APC can be carried out using
a dominant-negative Asef mutant for the binding. For example, an
Asef mutant that can bind to APC but does not exhibit GEF activity
can be used as an agent for inhibiting the binding of Asef to APC.
Such an Asef mutant can be obtained by designing mutants based on
the amino acid sequence of Asef and examining their binding activity
to APC according to a conventional method. More specifically, a
mutant that lacks the GEF domain of Asef can be exemplified. Alternatively,
a polypeptide consisting of the APC-binding region (amino acid sequence
from the 73.sup.rd to the 126.sup.th amino acid residue) in the
amino acid sequence of Asef is preferably used. A polypeptide that
inhibits the binding of Asef to APC that is selected from polypeptides
that are designed based on the amino acid sequence of this polypeptide,
can also be used. Further, inhibition of the binding of Asef to
APC can also be carried out by inhibiting the expression of the
APC gene. Inhibition of APC gene expression can be conducted by
using an oligonucleotide that exhibits an RNA interference effect
on the expression of the APC gene. Examples of the oligonucleotide
can include a cDNA having the nucleotide sequence set forth in SEQ
ID NO: 2 in the sequence listing. Further, the complementary RNA
(SEQ ID NO: 4 in the sequence listing) of the cDNA can also be used.
Alternatively, inhibition of the APC gene expression can be carried
out by using an antisense oligonucleotide against the APC gene.
The aforementioned oligonucleotide exhibiting an RNA interference
effect or the aforementioned antisense oligonucleotide can be obtained
from oligonucleotides that are designed on the basis of the nucleotide
sequence of the APC gene, by selecting oligonucleotides that specifically
inhibit the expression of APC using an APC gene expression system.
Inhibition of the GEF activity of Asef can be carried out, for
example, by using an inhibitor of GEF activity that can be identified
using Asef. Further, a compound that inhibits the expression of
the Asef gene or a compound that inhibits the binding of Asef to
APC may be identified using the Asef gene or using Asef and APC,
and the thus-identified compound may be used. An assay system for
identifying the compound can be constructed utilizing a known screening
system.
Metastasis of colorectal cancer can be inhibited by using an agent
for inhibiting Asef that contains the above-described substance
that inhibits the function and/or the expression of Asef as an active
ingredient. That is, an agent for inhibiting metastasis of colorectal
cancer comprising an agent for inhibiting Asef and a method for
inhibiting metastasis of colorectal cancer comprising using the
aforementioned agent for inhibiting Asef are also included in the
scope of the present invention. More specifically, an agent for
inhibiting metastasis of colorectal cancer comprising an oligonucleotide
having any one of the nucleotide sequences set forth in SEQ ID NOS:
1 to 4 in the sequence listing and a method for inhibiting metastasis
of colorectal cancer comprising using at least one of these oligonucleotides
can be exemplified.
Tumorigenicity and metastasis of colorectal cancer can be inhibited
by applying the agent for inhibiting metastasis of colorectal cancer
or the agent for inhibiting Asef of the present invention. More
specifically, the above described agent for inhibiting metastasis
of colorectal cancer or the agent for inhibiting Asef can be used
in the prevention and/or treatment of colorectal cancer and colorectal
cancer metastasis. From this viewpoint, an agent for preventing
and/or treating colorectal cancer comprising an effective amount
of the aforementioned agent for inhibiting metastasis of colorectal
cancer or the agent for inhibiting Asef as an active ingredient
are also included in the scope of the present invention. Further,
a method for preventing and/or treating colorectal cancer comprising
using the aforementioned agent for inhibiting metastasis of colorectal
cancer or the agent for inhibiting Asef can be also provided.
A pharmaceutical composition that includes the aforementioned agent
for inhibiting metastasis of colorectal cancer or the agent for
inhibiting Asef can be thus provided according to the present invention.
Suitable dosage ranges of the pharmaceutical composition of the
present invention can be can be determined according to the following:
effectiveness of the ingredients contained therein; the route of
administration; the properties of the prescription; the characteristics
of the symptoms of the subject; and the judgment of the physician
in charge. In general, a suitable dosage may fall, for example,
within a range of approximately 0.01 .mu.g to 100 mg per 1 kg of
the body weight of the subject, and preferably within a range of
approximately 0.1 .mu.g to 1 mg per 1 kg. However, a dosage may
be altered using conventional experiments for optimization of a
dosage that are well known in the art. The aforementioned dosage
can be divided for administration once to several times a day. Alternatively,
periodic administration once every few days or few weeks can be
employed.
When using an oligonucleotide that is capable of inhibiting the
expression of the Asef gene or APC gene, it is possible to produce
the oligonucleotide into the cell of the target by use of gene therapy.
The gene therapy can be performed by using a known method. For example,
a non-viral transfection comprising administering the oligonucleotide
directly by injection and a transfection using a virus vector can
both be applied. A method is recommended for non-viral transfection
that comprises administering a phospholipid vesicle such as a liposome
that contains the oligonucleotide, as well as a method comprising
administering the oligonucleotide directly by injection. A liposome
for use in this method can be more preferably exemplified by a cationic
liposome. A vector used for a transfection using a virus vector,
into which the oligonucleotide is incorporated, can be preferably
exemplified by a DNA virus vector such as a retrovirus vector, an
adenovirus vector, an adeno-associated virus vector and a vaccine
virus vector, or a RNA virus vector. Use of these virus vectors
enables administration to be carried out effectively. Further, a
method is recommended for a transfection using a virus vector that
comprises administering a phospholipid vesicle such as a liposome
that contains the vector.
A medicament of the present invention can be prepared as a medicament
that contains only an active ingredient of the agent for inhibiting
metastasis of colorectal cancer or the agent for inhibiting Asef,
but it is ordinarily prepared as a pharmaceutical composition using
one or more kinds of a pharmaceutical carrier.
The amount of the active ingredient contained in the pharmaceutical
formulation of the present invention can be appropriately selected
from a broad range. In general, a suitable amount may fall within
a range of approximately 0.00001 to 70 wt %, preferably approximately
0.0001 to 5 wt %.
Examples of the pharmaceutical carrier include a diluent or excipient
such as a filler, expander, binder, wetting agent, disintegrator,
surfactant, or lubricant that are normally used in accordance with
the form of use of the formulation, and these can be suitably selected
and used in accordance with the administration route of the formulation
to be obtained. Examples of the carrier include physiological saline,
buffered physiological saline, dextrose, water, glycerol, ethanol,
and mixtures thereof. A carrier is not limited to these examples,
and any substance can be used according to one's desire as long
as the substance can be used for formulation of a common medicament.
The pharmaceutical composition of the present invention can be
used as a solution formulation. It can also be used as a lyophilized
formulation in order to preserve it, which can be used by dissolving
it in water or in a buffered solution including physiological saline
or the like to prepare it to a suitable concentration just before
use.
When administering the pharmaceutical composition of the present
invention, the pharmaceutical composition may be used alone or may
be used together with other compounds or medicaments necessary for
the treatment.
In terms of a route of administration, it may be either systemic
administration or local administration. The route of administration
that is appropriate for a particular disease or symptomatic conditions
should be selected. As examples, parenteral administration including
normal intravenous injection, intraarterial administration, subcutaneous
administration, intracutaneous administration and intramuscular
administration can be employed. Oral administration can be also
employed. Further, transmucosal administration or dermal administration
can be employed. Direct administration to the neoplasm by an injection
or the like can be employed.
In terms of an administration form, various forms can be selected
from administration forms that are known to those skilled in the
art, and typical examples thereof include an administration form
of a solid formulation such as a tablet, pill, powder, powdered
drug, fine granule, granule, or capsule, as well as an administration
form of a liquid formulation such as an aqueous formulation, ethanol
formulation, suspension, fat emulsion, liposome formulation, clathrate
such as cyclodextrin, syrup, or an elixir. These can be further
classified according to the administration route into an oral formulation,
parenteral formulation (drip injection formulation or injection
formulation), nasal formulation, inhalant formulation, transvaginal
formulation, suppositional formulation, sublingual agents, eye drop
formulation, ear drop formulation, ointment formulation, cream formulation,
transdermal absorption formulation, transmucosal absorption formulation
and the like, which can be respectively blended, formed and prepared
according to conventional methods.
In general, when using the pharmaceutical composition of the present
invention for gene therapy, the pharmaceutical composition is preferably
prepared as an injection formulation, drip injection formulation
or liposome formulation. The pharmaceutical composition can also
be prepared in a form that allows administration thereof together
with a substance that enhances the efficiency of gene transfer,
such as protamine.
Powders, pills, capsules, and tablets can be prepared using an
excipient such as lactose, glucose, sucrose, or mannitol; a disintegrate
agent such as starch or sodium alginate; a lubricant such as magnesium
stearate or talc; a binder such as polyvinyl alcohol, hydroxypropyl
cellulose, or gelatin; a surfactant such as fatty acid ester; a
plasticizer such as glycerin, and the like. For preparation of a
tablet or a capsule, a pharmaceutical carrier in a solid state is
used.
A suspension can be prepared using water; sugars such as sucrose,
sorbitol, or fructose; glycols such as PEG; and oils.
Injectable solutions can be prepared using a carrier comprising
a salt solution, a glucose solution or a mixture of salt water and
a glucose solution.
Inclusion into a liposome formulation can be conducted in the following
manner: by dissolving the substance of interest in a solvent (e.g.,
ethanol) to make a solution, adding a solution of phospholipids
dissolved in an organic solvent (e.g., chloroform), removing the
solvent by evaporation and adding a phosphate buffer thereto, agitating
the solution and then subjecting it to sonication followed by centrifugation
to obtain a supernatant, and finally, filtrating the supernatant
to recover liposomes.
A fat emulsion can be prepared in the following manner: by mixing
the substance of interest, an oil ingredient (vegetable oil such
as soybean oil, sesame oil, olive oil, or MCT), an emulsifier (such
as a phospholipid), and the like; heating the mixture to make a
solution; adding water of a required quantity; and then emulsifying
or homogenizing by use of an emulsifier (a homogenizer, e.g., a
high pressure jet type, an ultrasonic type, or the like). The fat
emulsion may be also lyophilized. For conducting lipid-emulsification,
an auxiliary emulsifier may be added, and examples thereof include
glycerin or saccharides (e.g., glucose, sorbitol, fructose, etc.).
Inclusion into a cyclodextrin formulation may be carried out in
the following manner: by dissolving the substance of interest in
a solvent (e.g., ethanol); adding a solution of cyclodextrin dissolved
in water under heating thereto; chilling the solution and filtering
the precipitates; and drying under sterilization. At this time,
the cyclodextrin to be used may be appropriately selected from among
those having different void sizes (.alpha., .beta., or .gamma. type)
in accordance with the bulkiness of the substance of interest.
EXAMPLES
Hereinafter, the present invention may be explained more particularly
with examples; however, the present invention is not limited to
the following examples.
First, the following definitions relate to Asef, APC, and the mutants
thereof that are used in the examples herein. The proteins and the
mutants are referred to in abbreviated form.
Asef-full is a protein consisting of the wild-type, full-length
Asef. It was expressed as a haemagglutinin (HA)-tagged fusion protein
(HA-tagged wild-type Asef) or a Glutathione S-transferase (GST)
fusion protein (GST-Asef-full).
Asef-.DELTA.APC is a mutant that lacks the N-terminal APC-binding
region of Asef. This mutant possesses stronger GEF activity than
wild-type Asef.
Asef-.DELTA.DH is a mutant that lacks the DH domain (GEF domain)
of Asef. This mutant does not exhibit GEF activity.
Asef-ABR is a polypeptide consisting of the APC-binding region
(amino acid sequence from the 73.sup.rd to the 126.sup.th residue)
in the amino acid sequence of Asef. It was expressed as a maltose-binding
protein (MBP) fusion protein (MBP-Asef-ABR).
APC-arm is a polypeptide consisting of the armadillo repeat domain
of APC. It was expressed as a Myc-tagged fusion protein (Myc-tagged
APC-arm).
APC-876 is a polypeptide consisting of the consecutive amino acid
residues from the 1.sup.st (the N terminus) to the 876.sup.th residue
of the amino acid sequence of APC, which contains the armadillo
repeat domain.
APC-1309 is a polypeptide consisting of the consecutive amino acid
residues from the 1.sup.st (the N terminus) to the 1309.sup.th residue
of the amino acid sequence of APC, which contains the armadillo
repeat domain.
APC-876 and APC-1309 are truncated APC mutants that were identified
in colorectal tumors and familial adenomatous polyposis (FAP).
Adenoviruses that contain DNA encoding any of these proteins or
polypeptides were prepared by cloning polynucleotides that encode
each protein into the pAdeno-X adenoviral vector using the Adeno-X.TM.
Expression System (Clontech, Palo Alto, Calif.). The term "AdAsef-full"
hereunder refers to an adenovirus that contains DNA encoding Asef-full.
Adenoviruses that contain DNA encoding the other proteins or polypeptides
described above are likewise represented hereunder by adding "Ad"
to the designated name of each DNA.
Plasmids that contain DNA encoding any of the aforementioned proteins
or polypeptides were prepared according to a conventional method.
Cell culture and transfection of the aforementioned plasmids were
carried out as described in the following. MDCK cells (epithelial-like
cell line established from a normal canine kidney) and WiDr cells
were cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum (FCS). SW480 cells were cultured in Leibovitz's
L-15 medium supplemented with 10% FCS. DLD-1 cells and HCT15 cells
were cultured in RPMI 1640 medium supplemented with 10% FCS. HCT116
cells were cultured in McCoy's 5A medium supplemented with 10% FCS.
These cells were transfected with the above-described plasmids using
LipofectAMINE 2000 (Life Technologies, Carlsbad, Calif.).
Preparation and expression of proteins were carried out in the
following manner. Proteins fused to GST or MBP were synthesized
in Escherichia coli and isolated by absorption to glutathione Sepharose
(GSH-Sepharose; Pharmacia, Buchinghamshire, UK) or amylose resin
(New England Biolabs, Beverly, Mass.).
Short hairpin RNAs (hereunder, referred to in abbreviated form
as "shRNA"), such as shRNA-Asef and shRNA-APC used in
the RNA interference experiments were designed to inhibit the expression
of the Asef gene and the APC gene, respectively. The nucleotide
sequences of shRNA-Asef and shRNA-APC are set forth in SEQ ID NO:
1 and SEQ ID NO: 2 in the sequence listing, respectively. Further,
mutations were added to shRNA-Asef and shRNA-APC to prepare shRNAs
that did not inhibit the expression of the Asef gene and the APC
gene. These are mut-shRNA-Asef and mut-shRNA-APC, which are set
forth in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing,
respectively.
EXAMPLE 1
In order to examine the effects of Asef on cell-cell adhesion and
cell morphology, MDCK cells were infected with the above-described
adenoviruses. The adenoviruses used were AdAsef-full, AdAsef-.DELTA.APC,
AdAsef-.DELTA.DH and AdAPC-arm. AdLacZ was used as a control. Immunofluorescence
staining showed that the infection efficiency of the adenoviruses
to MDCK cells was 90% or more. Immunoblot analysis showed that each
of these viruses produces a protein of the expected size when infected
into MDCK cells.
Cell morphology was examined as follows. Cells were planted in
12-well tissue culture plates to get 3.0.times.10.sup.4 cells per
well. After 3 h of incubation at 37.degree. C., cells were infected
with adenoviruses (multiplicity of infection (MOI) =200), cultured
for a further 36 h and then observed by a phase-contrast microscope.
Cells infected with AdAsef-.DELTA.APC became flattened onto the
substratum and exhibited membrane ruffles and lamellipodia. In contrast,
cells infected with AdAsef-.DELTA.DH showed no morphological changes
and resembled uninfected cells.
Cell-cell adhesion was examined as follows. Infected cells were
scraped from plates in phosphate-buffered saline (PBS) containing
0.02% ethylenediamine tetraacetic acid (EDTA) and subjected to pipetting
20 times. The number of cell clusters (particles) was then counted.
The cell-cell adhesion was evaluated from a value obtained by dividing
the number of cell clusters by the total number of cells (Np/Nc).
When cells were dispersed by pipetting, cells infected with AdAsef-.DELTA.APC
dissociated efficiently, whereas uninfected cells and cells infected
with AdAsef-.DELTA.DH or AdLacZ remained as clusters (FIG. 1). These
results showed that Asef has a function to decrease cell-cell adhesion,
and that its GEF activity is essential for this function.
Further, immunohistochemical analysis using an anti-E-cadherin
antibody showed that overexpression of either AdAsef-full or AdAsef-.DELTA.APC
resulted in decreased amounts of E-cadherin localized at the sites
of cell-cell contact and enhanced amounts of E-cadherin localized
in the cytoplasm. Immunohistochemical analysis was conducted as
follows. After 36 h of adenovirus infection, MDCK cells were fixed
with 3.7% formaldehyde in PBS. The fixed cells were double-stained
with either a rat monoclonal antibody against E-cadherin (ECCD-2;
Calbiochem, San Diego, Calif.) and trimethylrhodamine isothiocyanate-conjugated
phalloidin (TRITC-conjugated phalloidin: Molecular Probes, Eugene
Oreg.), or the rat monoclonal antibody against E-cadherin and a
rabbit polyclonal antibody against .beta.-catenin (SantaCruz Biotechnology,
Santa Cruz, Calif.) for 60 min at room temperature. Staining patterns
obtained with anti-E-cadherin antibody and anti-.alpha.-catenin
antibody were visualized with fluorescein isothiocyanate-labelled
anti-rat IgG antibody and TRITC-labelled anti-rabbit IgG antibody,
respectively. The cells were photographed with a Carl Zeiss LSM510
laser scanning microscope. Staining with anti-.beta.-catenin antibody
showed a decreased amount of .beta.-catenin localized at the site
of cell-cell contact, although the decreased amount was not as prominent
as that of E-cadherin. In contrast, cells infected with AdAsef-.DELTA.DH
or AdLacZ did not show any change in localization of E-cadherin
or .beta.-catenin. These results suggest that GEF activity of Asef
is important for the changes in the localization of these molecules.
Immunoblot analysis of MDCK cell lysates showed that the total amount
of E-cadherin or .beta.-catenin did not change significantly upon
infection with AdAsef-full or AdAsef-.DELTA.APC. These results demonstrated
that the decrease in cell-cell adhesion resulting from the expression
of the Asef gene is due to a decrease in E-cadherin and .beta.-catenin
at the sites of cell-cell contact.
EXAMPLE 2
The effects of Asef on cell motility were examined using MDCK cells
that were made to express the Asef gene, the APC gene, or mutant
genes of these using the plasmids described above. Cell motility
was examined by cell migration assays using Transwell migration
chambers. The chambers used for MDCK cells were 12 mm in diameter
with a pore size of 12 .mu.m (Costar Corporation, Cambridge Mass.).
After 18 h of transfection, 3.0.times.10.sup.4 cells of MDCK cells
were added to the upper compartment of the chamber and allowed to
migrate toward the underside of the upper chamber for 18 h. Cell
migration was determined by measuring the cells that had migrated
to the lower side of the polycarbonate filters.
Cells infected with a plasmid that contains DNA encoding Asef-full
showed enhanced motility as compared with parental cells (MDCK)
or vector-transfected cells (Mock) (FIG. 2). Cells that were made
to co-express the Asef-full gene together with any one of the APC-arm
gene, the APC-876 gene and the APC-1309 gene were more motile than
cells transfected with the Asef-full gene alone. In the effect of
APC on the ability of Asef to promote cell-motility, APC-arm, APC-876
and APC-1309 were stronger than APC-full. In contrast, APC-arm alone
did not promote migration. In addition, cells transfected with the
Asef-.DELTA.APC gene showed a further enhanced migration reaction
as compared with cells cotransfected with the Asef-full gene and
the APC-arm gene. These results showed that Asef has the potential
to promote the migration of MDCK cells. It was shown that this potential
of Asef is further enhanced by APC, particularly a truncated APC
mutant that contains an armadillo repeat domain (APC-Arm). In addition,
Asef-.DELTA.DH did not promote the migration of MDCK cells, indicating
that the GEF activity of Asef is required for such migration stimulatory
activity.
Meanwhile, when the Asef-ABR gene was expressed together with the
APC-1309 gene, enhancement of cell migration was almost completely
inhibited. Asef-.DELTA.DH also inhibited APC-1309 mediated enhancement
of cell migration. These results indicate that the APC mutants,
APC-879 and APC-1309, which have been identified in colorectal cancers
and FAP, interact with Asef, and enhance its activity, thereby promoting
cell migration. On the other hand, when the full-length APC gene
was transfected into MDCK cells, no enhancement of Asef-induced
migration was observed (FIG. 2). This indicates that APC may not
be an efficient activator of Asef until APC is activated by truncation
in colorectal tumor cells.
Next, the motility of SW480 cells that are known to include Asef
and truncated APC mutants was examined. When SW480 cells were transfected
with plasmids that contain DNA encoding Asef-ABR, the migration
of the cells decreased to about 50% of that of the parental cells
or Mock cells (FIG. 2). Similarly, the migration of SW480 cells
transfected with Asef-.DELTA.DH plasmids decreased by to about 40%.
These results demonstrated that Asef-ABR or Asef-.DELTA.DH expressed
in cells acts on the binding of Asef to truncated APC mutants in
a dominant-negative manner, thereby inhibiting the cell migration.
EXAMPLE 3
The binding of Asef to truncated APC mutants in colorectal tumor
cells was examined as follows. First, 5.0.times.10.sup.6 cells of
SW480 cells were lysed in 500 .mu.l of buffer A (50 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 5 mM EDTA, 2 mM sodium vanadate (Na.sub.3VO.sub.4)
and 10 mM sodium fluoride) containing 1% Triton X-100. The lysate
was incubated with 2 .mu.g of anti-Asef antibody for 1 h at 4.degree.
C., and then the immunocomplex was adsorbed to protein G-Sepharose
6B for 2 h at 4.degree. C. After washing extensively with buffer
A containing 1% Triton X-100, the sample was resolved by SDS-PAGE,
and transferred to a polyvinylidene difluoride membrane filter (Immobilon
P; Millipore, Bedford, Mass.). The blot was analyzed by immunoblot
analysis using alkaline phosphatase-conjugated mouse anti-rabbit
IgG antibody (Promega, Madison, Wis.) as a second antibody. The
rabbit anti-Asef polyclonal antibody used was prepared by a conventional
method (Non-patent Reference 1).
The results showed that Asef co-immunoprecipitated with the truncated
APC mutant (FIG. 3a). Co-immunoprecipitation of Asef and the APC
mutant was inhibited by preincubation of the antibody with an excess
of the antigen for 2 h at 4.degree. C. These results demonstrate
that Asef is associated with APC mutants in SW480 cells.
Next, co-immunoprecipitation of GST-Asef-full and APC-arm was conducted
in vitro to examine the effect of Asef-ABR addition. First, APC-arm
was produced by in vitro translation (IVT-APC-arm), and incubated
with GST-Asef-full bound to Sepharose in the presence of MBP-Asef-ABR.
The relative amounts of APC-arm to MBP-Asef-ABR were varied as indicated
in FIG. 3b. APC-arm bound to GST-Asef-full-Sepharose was visualized
by SDS-PAGE followed by autoradiography (top panel of FIG. 3b).
MBP-Asef-ABR added to the reaction mixture was visualized by subjecting
the gel to Coomassie blue staining (bottom panel of FIG. 3b). The
results showed that the amount of co-immunoprecipitate of APC-arm
and GST-Asef-full decreased in a dose-dependent manner along with
the increasing amounts of Asef-ABR added. More specifically, it
was revealed that Asef-ABR inhibits the binding of Asef to the APC
mutant in vitro.
The inhibition of the migration of SW480 cells was achieved using
Asef-ABR that inhibits the binding of Asef to the APC mutant in
a dominant negative manner.
EXAMPLE 4
Various colorectal tumor cell lines were infected with adenoviruses
that contain DNA encoding Asef-full, Asef-.DELTA.APC or Asef-.DELTA.DH,
and assessed by cell migration assays in the same manner as in Example
2. The colorectal tumor cell lines used were SW480, DLD-1, HCT15,
WiDr and HCT116. SW480 cells, DLD-1 cells, HCT15 cells and WiDr
cells contain truncated APC mutants. HCT116 cells contain normal
APC but mutated .beta.-catenin.
The results are shown in FIG. 4. When SW480 cells were infected
with AdAsef-full or AdAsef-.DELTA.APC, their motility was enhanced.
Further, when SW480 cells, DLD-1 cells, HCT15 cells and WiDr cells
were infected with AdAsef-.DELTA.DH, their motility was partially
inhibited. In contrast, the motility of HCT116 cells was not inhibited
by AdAsef-.DELTA.DH. This indicates that full-length APC in HCT116
is unable to induce the activation of Asef. These results demonstrated
that the activation of Asef is induced in colorectal tumor cells
that contain truncated APC mutants, while the activation of Asef
is not or is hardly induced in cells that contain normal APC. The
results also showed that the activation is inhibited by Asef-.DELTA.DH.
EXAMPLE 5
The interaction of Asef with APC mutants in the migration of colorectal
tumor cells was investigated using RNA interference experiments.
The experiments were carried out using the pSHAG-1 vector system
(Non-patent Reference 13). The colorectal tumor cell lines used
were SW480 cells, WiDr cells, LS180 cells and HCT116 cells. SW480
cells and WiDr cells contain truncated APC mutants. LS180 cells
and HCT116 cells contain normal APC but mutated .beta.-catenin.
Cell migration assays were carried out in the same manner as in
Example 2 to assess various colorectal tumor cells transfected with
expression vectors that contain either shRNA-Asef or shRNA-APC.
The results showed that SW480 cells and WiDr cells that were transfected
with either shRNA-Asef or shRNA-APC exhibited decreased motility
as compared with cells that were transfected with mut-shRNA-Asef
or mut-shRNA-APC (FIG. 5). In contrast, this phenomenon was not
observed in LS180 cells and HCT116 cells.
Next, immunoblot analysis was carried out in the same manner as
in Example 3 to assess cells transfected with expression vectors
that contain oligonucleotides encoding any one of shRNA-Asef, shRNA-APC,
mut-shRNA-Asef and mut-shRNA-APC. Meanwhile, changes in .alpha.-tubulin
were measured as a control. The results showed that shRNA-Asef and
shRNA-APC almost completely inhibited the expression of the Asef
gene and the APC gene, respectively.
It was thus demonstrated that the motility of colorectal tumor
cells that contain truncated APC mutants is decreased by inhibiting
the expression of the Asef gene or the APC gene. More specifically,
the results indicated that interaction of Asef with APC mutants
plays an important role in the migration of colorectal tumor cells.
EXAMPLE 6
Cells prepared by making the Asef dominant-negative mutant Asef-ABR
express in human SW480 colorectal tumor cells were respectively
transplanted to SCID mice to observe changes in their tumorigenicity
or proliferation. Asef-ABR plasmids were transfected into SW480
colorectal tumor cells by lipofection. The thus-obtained 3 clones
were respectively cultured in vitro using L-15 medium containing
G418 at a final concentration of 1 mg/ml, and then transplanted
subcutaneously in the flanks of 8-week-old SCID mice at 1.times.10.sup.7
cells/0.1 ml/mouse (2 to 4 individuals per group). Twenty days after
transplanting the tumor cells, tumor lumps were excised to measure
their weights. The weight of tumor lumps (T) of each mouse that
was transplanted with the clone was divided by the value of a control
group (C) to get an inhibition ratio (abbreviated as "IR")
that was expressed as a percentage [IR (%)=T/C.times.100]. The expression
of Asef-ABR in the transplanted cells was confirmed by a conventional
method.
Decreased tumorigenicity or delayed proliferation was seen in 2
out of 3 clones stably expressing Asef-ABR alone (Table 1). Thus,
it is believed that Asef participates in tumorigenicity or tumor
cell proliferation.
TABLE-US-00001 TABLE 1 number of mice with tumor/ group weight
.+-. SD (g) IR (%) transplanted with tumor SW480 0.496 .+-. 0.080
0 4/4 ABR-2 0.609 .+-. 0.069 -22.7 3/3 ABR-8 0.000 .+-. 0.000 100
0/3 ABR-17 0.203 .+-. 0.056 59.1 4/4
EXAMPLE 7
Asef-ABR clones (see Example 6) that were prepared using the human
HT29 colorectal tumor cell line were transplanted into SCID mice
to observe changes in their tumorigenicity or proliferation. 15
.mu.g of Asef-ABR plasmids were transfected into 5.times.10.sup.6
cells of HT29 cells by lipofection. The thus-obtained 5 clones were
respectively cultured in vitro using DMEM medium containing G418
at a final concentration of 1 mg/ml, and then transplanted into
the spleen of 8-week-old SCID mice at 1.times.10.sup.6 cells/0.05
ml/mouse (4 individuals per group). Eighteen days after transplanting
the tumor cells, the mice were injected with ink via tail vein,
and then sacrificed with bleeding under anesthesia with ether. The
spleen and the liver were excised to measure their weights.
Tumor formation in the spleen and the liver was not observed in
4 out of 5 clones that are the stable transfectant expressing Asef-ABR
dominant-negative mutant, which were prepared using HT29 cells (Table
2). The same phenomenon was also observed in 3 clones prepared in
Example 6 using SW480 colorectal tumor cells. Thus, it was demonstrated
that Asef participates in tumorigenicity and tumor cell proliferation.
Further, no tumor formation in the liver was observed when using
the stable transfectant expressing Asef-ABR dominant-negative mutant,
indicating that Asef-ABR dominant-negative mutants inhibit tumor
metastasis.
TABLE-US-00002 TABLE 2 group liver weight .+-. SD (g) spleen weight
.+-. SD (mg) normal 1.340 .+-. 0.176 30.8 .+-. 7.1 parental cell
line 2.236 .+-. 0.153 124.5 .+-. 20.4 (HT29) Asef-ABR-A7 1.584 .+-.
0.093 38.0 .+-. 3.9 Asef-ABR-B5 2.627 .+-. 0.392 129.3 .+-. 13.5
Asef-ABR-C3 1.579 .+-. 0.124 44.3 .+-. 11.0 Asef-ABR-C12 1.526 .+-.
0.070 37.8 .+-. 5.1 Asef-ABR-D11 1.359 .+-. 0.173 37.3 .+-. 4.6
INDUSTRIAL APPLICABILITY
In the present invention, it was found that Asef enhances the motility
of cells, and that it decreases cell-cell adhesion, and that this
function of Asef is activated by the gene product of the tumor suppressor
gene APC. Further, it was found that Asef enhances the motility
of the colorectal tumor cells and participates in the tumorigenicity
and metastasis thereof in colorectal cancers, particularly in colorectal
cancers in which mutant APCs are observed. According to the present
invention based on these findings, the agent for inhibiting metastasis
of colorectal cancer and the method for inhibiting metastasis of
colorectal cancer, which inhibit the function of Asef and/or inhibit
the expression of Asef gene, were provided. These inventions have
a significant effect for the prevention and/or treatment of colorectal
cancer and colorectal cancer metastasis.
SEQUENCE LISTING FREE TEXT
SEQ ID NO: 1: Designed oligonucleotide based on the nucleotide
sequence of human Asef to inhibit the expression of the Asef gene.
SEQ ID NO: 2: Designed oligonucleotide based on the nucleotide
sequence of human APC to inhibit the expression of the APC gene.
SEQ ID NO: 3: Designed oligonucleotide based on the nucleotide sequence
of human Asef to inhibit the expression of the Asef gene. SEQ ID
NO: 4: Designed oligonucleotide based on the nucleotide sequence
of human APC to inhibit the expression of the APC gene. SEQ ID NO:
5: Designed oligonucleotide based on the nucleotide sequence set
forth in SEQ ID NO: 1. SEQ ID NO: 6: Designed oligonucleotide based
on the nucleotide sequence set forth in SEQ ID NO: 2.
>
6 A Artificial Designed oligonucleotide based on the nucleotide
sequence of human Asef to inhibit the expression of the Asef gene
gactt ccagatctac tcggagtact g 3DNA Artificial Designed oligonucleotide
based on the nucleotide sequence of human APC to inhibit the expression
of the APC gene 2 aactgaggca tctaatatga aggaagtact t 3RNA Artificial
Designed oligonucleotide based on the nucleotide sequence of human
Asef to inhibit the expression of the Asef gene 3 uucggcugaa ggucuagaug
agccucauga c 3RNA Artificial Designed oligonucleotide based on the
nucleotide sequence of human APC to inhibit the expression of the
APC gene 4 uugacuccgu agauuauacu uccuucauga a 3DNA Artificial Designed
oligonucleotide based on the nucleotide sequence set forth in SEQ
ID NO acgactt ccaaatctac tcagagtact g 3DNA Artificial Designed oligonucleotide
based on the nucleotide sequence set forth in SEQ ID NO 2 6 aactaaggca
tataatatga aggaaatact t 3BR> |