WO2008051048A2

WO2008051048A2 - Method and materials for inhibiting a nuclear export of gsk3

Info

Publication number: WO2008051048A2
Application number: PCT/KR2007/005313
Authority: WO
Inventors: Jeong Moon Kim; Jung Kook Kim; Tae Han Kim; Jong Suk Lee; Jong In Yook
Original assignee: Jeong Moon Kim; Jung Kook Kim; Tae Han Kim; Jong Suk Lee; Jong In Yook
Priority date: 2006-10-27
Filing date: 2007-10-26
Publication date: 2008-05-02
Also published as: KR20080038047A

Description

METHOD AND MATERIALS FOR INHIBITING A NUCLEAR EXPORT OF GSK 3

TECHNICAL FIELD

The present invention relates to a method and material for inhibiting export of glycogen synthase kinase 3 (GSK3) from nucleus to cytoplasm, and more particularly to a polypeptide for inhibiting the export of GSK3 from nucleus to cytoplasm, which comprises (i) a glycogen synthase kinase 3 (GSK3)-binding domain (GBD) or (ii) an Axin-binding domain (ABD), and to a method for inhibiting a decrease in the concentration of GSK3, which is caused by the nuclear export of GSK3 through binding to Axin.

BACKGROUND ART

Glycogen synthase kinase-3 (GSK3) is serine/threonine kinase, which has a molecular weight of about 47 kD and phosphorylates about 50 proteins. GSK3 includes two isoforms, GSK3α and GSK3/3, which are known to be very similar and to have similar functions (Double, B. W. & Woodgett, J. R., J. Cell ScL, 116: 1 175, 2003).

GSK3 functions to regulate the phosphorylation of many proteins, such as β- catenin or Snail, during the pathological processes of immune and chronic inflammatory diseases, such as (1) individual development, (2) cancer development and progression, and (3) rheumatic arthritis. That is, an abnormality in signaling pathways, such as Wnt signaling, inhibits GSK3 phosphorylation of factors for cancer development and activation of metastasis, such as j8-catenin and Snail, so as to increase the half-life of the factors, thus promoting the development and progression of various diseases.

Thus, a material for promoting the intracellular or intranuclear activity of GSK3 will assist in inhibiting the development and progression of diseases, caused by Wnt signaling. In particular, if the activity of GSK3 is increased, the inhibition of GSK3 in the nucleus, caused by Wnt signaling or the like, can be reduced, and the development and progression of many diseases, caused by Wnt signaling, can be effectively inhibited.

However, an exact process for regulating the phosphorylation of substrate proteins, caused by GSK3, is not known yet, and a therapeutic agent against diseases caused by the inhibition of GSK3 activity in the nucleus is also not known yet. This is because mechanisms about the effect of Wnt signaling on GSK3 are not clearly found. In the present invention, the mechanism of the nuclear export function (function of exporting GSK3α and GSK3/3 from the nucleus to the cytoplasm) of GSK by Axin in the Wnt signaling pathway associated directly with such diseases was clearly identified, and thus a method either for inhibiting a decrease in the concentration or activity of GSK3 in the nucleus or for maintaining and increasing the concentration or activity thereof was suggested for the first time. Accordingly, a method and a therapeutic agent, which can treat diseases associated with the nuclear export of GSK3, could also be suggested.

An important mechanism identified in the present invention is that a GSK3 regulatory factor, which binds to GSK3 so as to perform the nuclear export of

GSK3, is Axin in the Wnt signaling pathway. The Axin gene have two isoforms,

Axinl and Axin2 (conductin). It is known that the Axinl and Axin2 genes regulate the phosphorylation of proteins through different transcriptional regulatory processes. The Axin genes promote GSK3 phosphorylation of β- catenin so as to reduce the half life of the protein, thus serve as negative regulators of Wnt signaling (US 2001/0052137A1).

In addition, there are FRAT-I and FRAT-2 genes, which bind to GSK3 so as to perform the nuclear export of GSK3. However, unlike Axin known to promote the phosphorylation of /3-catenin, FRAT was found to be an inhibitor of GSK3 (Ciani, L. et al., J. Cell Biol., 164:243, 2004; Yost, C. et al, Cell, 93: 1031, 1998), and in addition, it is different from GSK.3 with respect to binding points. In particular, it is still unclear what the role of the FRAT gene is in the Wnt signaling pathway, even though it can not be said that the FRAT gene has no connection with Wnt, because, if one of the Axin-1 and Axin-2 genes is knocked out, normal development will not occur (Chia, IV & Costatini, F., MoI. Cell Biol., 25:4371, 2005), however, if the FRAT-I, -2 and -3 are all knocked out, normal development is possible (van Amerongen, R. et al., Genes De., 19:425, 2005).

The Axin gene reduces the concentration of GSK3 through the nuclear export function of exporting GSK3α and GSK3/3 from the nucleus to the cytoplasm, thus inhibiting the phosphorylation of nuclear transcriptional regulatory proteins such as Snail. Particularly, Wnt signaling increases the transcription and activation of Axin2 via /3-catenin, and thus reduces the concentration of GSK3 in the nucleus through the GSK3 nuclear export function of Axin2. That is, the activation of /3-catenin by the Wnt signaling reduces the concentration of GSK3 in the nucleus through the activation of Axin2, which performs the GSK3 nuclear export function, thus inhibiting the phosphorylation of various regulatory factors in the nucleus.

Accordingly, on the basis of the GSK3 nuclear export function of Axin, newly identified in the present invention, when the activity of /3-catenin that contributes to the activation of Axin is inhibited or when the Wnt signaling in the Wnt//3-catenin pathway, which is involved directly in the activity of /3-catenin, is inhibited, it is possible to prevent export of GSK from nucleus to cytoplasm, such that it is possible to prevent and treat various diseases, which are developed and progressed due to the Wnt signaling.

Although many drugs for inhibiting the activity of GSK3 have been developed to date, a concept and material of increasing the activity of GSK3 to inhibit the onset and progression of diseases, caused by the Wnt signaling, are not yet known. On the basis of newly identified the GSK3 nuclear export function of Axin, the present invention suggests a concrete method and materials for inhibiting the onset and progression of diseases, caused by Wnt signaling. That is, because many diseases occurring via /3-catenin is attributable to an abnormality in the concentration of GSK3 in the nucleus, caused by Axin in the Wnt signaling pathway, if a decrease in the concentration of GSK3 in the nucleus is inhibited using the nuclear export function of jS-catenin and Axin, it is possible to develop new concepts, methods and materials for treating cancer development and metastasis and various chronic immune diseases.

In order for Axin to perform the nuclear export of GSK3, a close structural contact between Axin and GSK3 is essential. According to the already known Axin-GSK3 binding structure, the domain of amino acids 383-401 of Axinl or the domain of amino acids 365-385 of Axin2 forms α-helix, and the hydrophobic residues in the center thereof bind to a groove consisting of the helix (amino acids 262-273) and extended loop (amino acids 285-299) of GSK3/5.

Meanwhile, a protein transduction domain (PTD), which can effectively deliver proteins into cells is known (Joliot, A. et al, Nature Cell Biol., 6: 198, 2004; US

2006/0222657A1). Also, haemagglutinin2 (HA2 domain), which is an influenza membrane fusion protein capable of avoiding a decrease in biological activity, caused by macropinosome or endosome, was also found (Skehel, JJ. et al.,

Biochem. Society Med., 10:310, 2004; Jehangir, S. W. et al., Nature Medicine, 10:310, 2004; Loredana Vaccaro et al., Biophysical J., 88:25, 2005). Using this technology, the intracellular delivery of various peptides, the biological activities of which can be expected, has been attempted. However, it is an initial stage where the probability of delivering peptides into cells was proved, and only the possibility thereof was confirmed but still a method or materials of treating diseases using it has not been suggested. This is because it is difficult to predict how materials involved in various human diseases interact with each other to develop diseases, and in addition, obtaining experimental results while precisely controlling many exceptional events is work, which is extremely difficult and requires creative insight.

In the present invention, the nuclear export function of GSK3 by Axin in the Wnt signaling pathway, and the fact that the nuclear export function causes diseases, were found for the first time, and on the basis of these findings, the above- described prior technologies were interactively combined with each other to suggest an entirely new method and materials for treating diseases.

Accordingly, the present inventors have made many efforts to develop a method and materials for treating diseases, which occur due to a reduction in the concentration of GSK in the nucleus, and, as a result, have found that a polypeptide, comprising a GSK3 (glycogen synthase kinase 3)-binding domain (GBD) or an Axin-binding domain and a PTD, which enables the domain to permeate through the cell membrane without the aid of a cell membrane receptor, is useful for treatment of various diseases, which are caused due to a decrease in the concentration of GSK3 in the nucleus, thereby completing the present invention.

SUMMARY OF THE INVENTION

It is a main object of the present invention to provide a polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, caused by GSK3 binding to Axin. Another object of the present invention is to provide a recombinant vector for expressing said polypeptide, recombinant microorganisms transformed with said recombinant vector, and a method for preparing said polypeptide inhibiting the export of GSK3, caused by GSK3 binding to Axin, the method comprising culturing said recombinant microorganisms.

Still another object of the present invention is to provide a pharmaceutical composition for inhibiting the growth and metastasis of cancer cells, and a pharmaceutical composition for treating immune diseases, each of the compositions containing said polypeptide as an active ingredient.

Yet another object of the present invention is to provide a method for inhibiting a decrease in the concentration of GSK3, which is caused by the nuclear export of GSK3 through GSK3 binding to Axin.

To achieve the above objects, in one aspect, the present invention provides a polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, which comprises: (a) a protein transduction domain (PTD), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; and (b) a GSK3 (glycogen synthase kinase 3)-binding domain (GBD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to GSK3.

In another aspect, the present invention provides a polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, which comprises (a) a protein transduction domain (PTD), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; and (b) an Axin-binding domain (ABD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to Axin. The polypeptides according to the present invention may additionally comprise (c) a HA2 domain which can avoid a decrease in biological activity, which is caused by macropinosome or endosome. Also, the polypeptides may additionally comprise a nuclear localization signal (NLS) for inhibiting export of GSK3 from nucleus to cytoplasm.

In the present invention, the PTD is preferably TAT, the GBD is preferably an Axin-derived, GSK3-binding domain, which inhibits the binding of GSK3 to Axin, and the GBD is preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7.

In the present invention, the ABD is preferably a GSK3 -derived, Axin-binding domain, which inhibits the binding of GSK3 to Axin.

In still another aspect, the present invention provides a recombinant vector, comprising: a base sequence encoding a protein transduction domain (PTD), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; and a base sequence encoding a GSK3 (glycogen synthase kinase 3)-binding domain (GBD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to GSK3.

In yet another aspect, the present invention provides a recombinant vector, comprising: a base sequence encoding a protein transduction domain (PTD), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; and a base sequence encoding an Axin-binding domain (ABD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to Axin.

The recombinant vectors according to the present invention may additionally comprise a base sequence encoding a HA2 domain which can avoid a decrease in biological activity, caused by macropinosome or endosome. Also, the recombinant vectors may additionally comprise a base sequence encoding a nuclear localization signal (NLS) for inhibiting the export of GSK3 from nucleus to cytoplasm.

In still another aspect, the present invention provides recombinant microorganisms transformed with said recombinant vector, and a method for preparing a polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, the method comprising culturing said recombinant microorganisms.

In still another aspect, the present invention provides a pharmaceutical composition for inhibiting the growth and metastasis of cancer cells, the composition comprising said polypeptide as an active ingredient.

In still another aspect, the present invention provides a pharmaceutical composition for treating immune diseases, which contains said polypeptide as an active ingredient. In the present invention, said immune disease is preferably arthritis.

In yet another aspect, the present invention provides a formulation for treating diseases, which occurs due to a decrease in the concentration of GSK3 in the nucleus, the formulation comprising said polypeptide bound to nano-sized materials or quantum dots (Q-dots).

In yet another aspect, the present invention provides a gene therapy agent, containing: a therapeutic gene encoding said polypeptide; and a carrier thereof. In the present invention, the carrier is preferably a viral vector.

In still another aspect, the present invention provides a diagnostic agent for diagnosing diseases, which occur due to a decrease in the concentration of GSK3 in the nucleus, the diagnostic agent comprising said polypeptide bound to nano- sized particles or quantum dots (Q-dots).

In the present invention, the diseases occurring in a decrease in the concentration of GSK3 in the nucleus is preferably cancers or immune diseases.

In yet another aspect, the present invention provides a method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, the method comprising: (a) blocking the Axin-binding domain of GSK3; (b) blocking the GSK3-binding domain of Axin; (c) inhibiting the activation of Axin; or (d) increasing the expression of GSK3 in the nucleus.

In the present invention, blocking the Axin-binding domain of GSK3 can be performed using a compound, a peptide or a polypeptide, which bind to the Axin- binding domain of GSK3. Also, blocking the GSK3-binding domain of Axin can be performed using a compound, a peptide or a polypeptide, which bind to the GSK3-binding domain of Axin. Also, inhibiting the activation of Axin can be performed by inhibiting Wnt signaling in a Wnt//3-catenin pathway or inhibiting the activity of /3-catenin.

Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of the inventive cell-permeable polypeptide to inhibit the nuclear export of GSK3 using the GSK3-binding domain of Axin2 and to increase the concentration of GSK3 in the nucleus (A: a cell-permeable polypeptide for inhibiting Axin-GSK3 binding; and B: a recombinant vector for preparing the inventive polypeptide).

FIG. 2 is a schematic diagram showing the activation of β-catenin, an increase in Axin2 expression and the regulation of GSK3 in the nucleus, which result from Wnt signaling.

FIG. 3 is a schematic diagram showing a process of regulating the concentration of GSK3 in the nucleus by Wnt signaling.

FIG. 4 is a schematic diagram showing an Axin-GSK3 binding structure.

FIG. 5 shows the effect of Wnt signaling on the expression of E-cadherin and β- catenin.

FIG. 6 shows the effects of Wnt signaling and a Snail gene on the activity and metastasis of E-cadherin (A: the effect of Wnt signaling on the expression of E- cadherin, /3-catenin and Snail genes; B: the effect of the Snail gene on the activity of an E-cadherin promoter; C: the effect of Wnt signaling on metastasis; and D: the effect of the Snail gene on metastasis).

FIG. 7 shows the effects of /3-catenin on metastasis and the activation of an E- cadherin promoter ("A" shows that, only by the activation of /3-catenin, the invasive growth and metastasis of cancer cells are promoted, and the activation of the E-cadherin promoter is reduced, and "B" shows that, when a dominant- negative TCF (DN-TCF) expression vector is introduced into MCF-7/Wnt cells, metastasis does not occur).

FIG. 8 shows the effects of /3-catenin on the expression of a Snail gene and E- cadherin (A: the Topflash activity of transformants, into which Mock, Snail and S33Y /3-catenin have been introduced, respectively; B: the effect of /3-catenin on the expression of the Snail gene; C: the effect of a /3-catenin-TCF flag on the expression of the Snail gene; and D: the effect of /3-catenin on the expression of the Snail gene).

FIG. 9 shows the effect of /3-catenin on the expression of E-cadherin and the metastasis of the Snail gene ("A" shows that the activation of /3-catenin reduces the expression of E-cadherin through the Snail gene; and "B" shows that metastasis does not occur in MCF-7/S33Y Snail siRNA cells).

FIG. 10 shows the effects of Axin2 on the expression and metastasis of Snail (A: the effect of /5-catenin on the expression of Axin2; B: the effect of Axin2 on the expression of Snail; C: the effect of Axin2 on the half-life of Snail; and D: the effect of Axin2 on metastasis).

FIG. 1 1 shows the effects of Axin2 on GSK3/3 (A: the effect of Axin2 on the location of GSK3/3; B: the effect of Axin2 on a reduction in the expression of GSK3/3 in the nucleus; and C: the correlation between GSK3/3 and Snail expression).

FIG. 12 shows the mechanisms of Axin2 acting on GSK3 (A: a nuclear export signal (NES) in the Axin2 gene; B: shows that the NES functions to export GSK3 from the nucleus to the cytoplasm; and C: the effect of Axin2 on the activation of GSK3 and E-cadherin promoters).

FIG. 13 shows that the activation of /3-catenin induces the expression of Axin2 and that Axin2 exports GSK3 from the nucleus to increase the expression of Snail.

FIG. 14 shows increases in the expressions of Axin2 mRNA and Snail in human breast cancer tissue. FIG. 15 shows increases in the expression of Axin2 and Snail in cancer cells metastasized from human breast cancer tissue into lymphatic blood vessels and in cancer tissues metastasized into lymph nodes.

FIG. 16 shows that an increase in the expression of Axin2 in human cervical cancer cells leads to a decrease in the concentration of GSK3 in the nucleus and an increase in the expression of Snail.

FIG. 17 shows increase in the expression of Wntl mRNA in human cervical normal, malignant lesion and invasive cancer.

FIG. 18 shows increase in the expression of Wnt3a mRNA in human cervical normal, malignant lesion and invasive cancer.

FIG. 19 shows increase in the expression of Axin2 mRNA in human cervical normal, malignant lesion and invasive cancer.

FIG. 20 shows increase in the expression of Snail in human cervical normal, malignant lesion and invasive cancer.

FIG. 21 shows that, when the Axin2 gene and Snail gene in cervical cancer cells are knocked out, the invasive growth and metastasis of the cancer cells are blocked.

FIG. 22 shows that the induction of expression of Wntl or Wnt3a leads to a decrease in the expression of GSK3α and GSK3/3 in the nucleus.

FIG. 23 is a schematic diagram for constructing an Axin2-derrived GSK3 binding polypeptide and an expression vector. FIG. 24 is a schematic diagram for constructing a GSK3-derived Axin binding polypeptide and an expression vector.

FIG. 25 is a schematic diagram showing that the use of nanobeads leads to an increase in the expression of GSK3 in the nucleus.

FIG. 26 shows real-time cell images obtained by binding each of a cell permeable peptide, a HA2 domain and a GSK3 binding domain to nanobeads, and then delivering the resulting structures into cells.

FIG. 27 shows that a polypeptide comprising a nuclear localization signal bound to GFP (green fluorescent protein) is imported specifically into the nucleus.

DETAILED DESCRIPTION OF THE INVENTION, AND

PPRFERRED EMBODIMENTS

In the present invention, a mechanism leading to a series of signaling pathways, that is, Wnt signaling, /3-catenin activation and an increase in the expression of an Axin2 gene, was identified. On the basis of the mechanism, the cytological mechanism of signaling pathways, in which GSK3 in the nucleus is exported to the cytoplasm according to the GSK3 nuclear export function, resulting in a decrease in the concentration of GSK3 in the nucleus, was found for the first time. On the basis of this finding, a fundamental concept for the treatment of various diseases caused by a decrease in the concentration of GSK3 was developed.

Accordingly, the present invention relates to a method and materials for inhibiting export of GSK from nucleus to cytoplasm, caused by the GSK3 nuclear export function, through binding to GSK competitively with Axin. That is, the present invention relates to a polypeptide capable of effectively inhibiting the nuclear export of GSK3, in which the polypeptide performs the same function as that of Axin2 and binds to GSK3, but has little or no effect on the function of GSK3.

In the following description of the present invention, the theory of the present invention will be logically described using many prior articles. However, the development of this logic is based on selection from among innumerable prior articles through a considerable creative insight and study process (among the prior articles, there are many articles showing contradictory results, and in this case, there is a need of a process of selecting and finding reasonable articles and theories through more extensive studies or experiments), and thus is considered to be obtained through a very meaningful process, even though it is in connection with the results in the prior articles.

Although the binding domains of Axin and GSK3 are structurally known (Dajani et ai, EMBO J., 22:494, 2003), the GSK3 nuclear export function of Axin, is not known yet. It was found that the domain of amino acids 383-401 of Axinl , which is known to be a domain binding to GSK3, binds to a groove formed of the helix (amino acids 262-273) and extended loop (amino acids 285-299) of GSK3/3. Particularly, a hydrophobic ridge consisting of phenylalanine 388 (Phe388), leucine 392 (Leu392), leucine 396 (Leu396) and valine 399 (Val399) plays a decisive role in binding to GSK3/3. Axinl and Axin2 have very similar structures and are also similar with respect to the function of exporting GSK3 from the nucleus to the cytoplasm. Furthermore, it is inferred that Axinl and Axin2 have the same function, that is, very similar structures, because normal development is progressed, even when Axinl and Axin2 are knocked out during the developmental process (Chia, LV. & Costantini, F., MoI. Cell Biol, 25:4371 , 2005).

In the present invention, it was found through many trials and errors that, when the expression of Axinl or Axin2 in cells was induced, the expression of GSK3 in the nucleus would be rapidly reduced, and thus the expression of the Snail protein that is phosphorylated and degraded by GSK3 would be strongly increased.

Meanwhile, it is known that the FRAT gene has three isoforms, FRAT- 1 , -2 and - 3, which also have the nuclear export function of exporting GSK from the nucleus to the cytoplasm (Franca-Koh, J. et al, J. Biol. Chem., 277:43844, 2002). Although there can be a slight similarity in binding structures, FRAT and Axin are different with respect to not only their structures, but also their binding points and binding patterns. Particularly, although it is not considered that FRAT has no connection with Wnt, FRAT is significantly different from Axin, which is the component of the Wnt signaling pathway, because a normal developmental process is progressed, even when FRAT-I, -2 and -3 are all knocked-out (Dajani et al, EMBO J., 22:494, 2003; van Amerongen et al, Genes Dev., 19:425, 2005).

The present invention has a concept fundamentally different from the prior development of various compounds for inhibiting phosphorylating enzymes. That is, the prior compounds for inhibiting phosphorylating enzymes are mostly drugs binding specifically to an ATP (adenosin triphosphate)-binding kinase domain. However, the polypeptides disclosed in the present invention is fundamentally conceptually different from the prior inhibition of phosphorylating enzymes, in that it does not inhibit the kinase domain, but rather inhibits the GSK3 nuclear export function of Axin, thus increasing the kinase activity of GSK3 in the nucleus.

In one aspect, the present invention relates to a cell-permeable polypeptide for inhibiting the nuclear export of GSK3 and a compound having a function similar thereto, which comprise: a PTD (protein transduction domain), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; a GSK3 (glycogen synthase kinase 3)-binding domain (GBD), which binds to GSK3 to inhibit the nuclear export (from nucleus to cytoplasm) of GSK3; a HA2 domain (HD) capable of avoiding a decrease in biological activity, caused by macropinosome or endosome; and a nuclear localization signal, which enables the polypeptide to be easily imported into the nucleus so as to effectively inhibit the GSK3 nuclear export function of Axin.

As shown in FIG. IA, a preferred embodiment of the polypeptide according to the present invention consists of a cell-permeable tat as PTD, a HA2 (H A2 endosomal rescue signal) for macropinosome rescue, and a GSK3 binding domain (the sequence of amino acids 370-390 of hAxin2). In order to increase the concentration of GSK3 in the nucleus, it is preferable to use the cell permeable polypeptide, but the scope of the present invention is not limited thereto. For example, it is also possible to use a compound, which can bind to GSK3 competitively with Axin so as to inhibit Axin-GSK3 binding, thus increasing the expression and concentration of GSK3 in the nucleus. In addition, it is also possible to substitute an expression vector, descried in Example 10, with a viral expression vector such as adenovirus or retrovirus, for use in gene therapy.

In the present invention, TAT(RKKRRQRRR) is used as the PTD, but the scope of the present invention is not limited thereto. For example, AntHD (drosophila homeoprotein atennapedia transcription protein), VP22 (virus protein22) peptide and mph-1-btm (mouse transcriptional inhibitory factor- 1-biomolecule transduction mortif), Penetratin, Buforin II, Transportan, Ku70, Prion, pVEC, Pep-1 , PTD-5, KALA (Joliot, A. et al, Nature Cell Biol, 6: 198, 2004; Kabouridis, P. S., Trends Biotechnol, 21 :498, 2003) or the like may also be used instead of TAT (transactivator of transcription).

In the present invention, in order to overcome the case where the cell permeable peptide permeates through the cell membrane so as to be restricted inside endosomes or macropinosomes, an influenza virus-derived HA2 peptide (Jehangir, S.W. et al., Nature Med., 10:310, 2004) is used, but the scope of the present invention is limited thereto. For example, it is possible to use drugs or peptides such as cloroquine or sucrose which can avoid the embedding of the cell permeable peptide in a lipid bilayer.

The haemagglutinin (HA) of influenza is a kind of glycoprotein, which is the component of a viral envelope, and serves to mediate the adhesion of virus to target cells or the fusion of the target cell membrane with the viral envelope membrane. In the case of general viral infection, virus attached to the cell surface enters an endosome, in which it is exposed to a relatively low pH. This change in pH leads to not only a morphological change in which the amino terminal end of HA is much more exposed, but also the fusion between the viral envelop and the endosomal membrane.

HA consists of two polypeptide fragments, HAl and HA2, in which the HAl fragment forms a sialic acid-binding domain so as to mediate adhesion to the host cell surface. The HA2 fragment forms a membrane-spanning anchor, and the amino-terminal domain acts in a fusion reaction mechanism.

Haemagglutinin2 (HA2 domain) has one or more T-helper cell recognition site, but has no B-cell recognition site. Thus, it induces a T-dependent immunological response to an antigen bound to the HA2 domain, but does not induce an antibody response to itself. The HA2 subunit includes a hydrophobic amino acid sequence close to the carboxyl terminal end generally extended through the lipid outer membrane of virus, and thus it is suitable as a helper peptide, which can promote the binding of liposome to the lipid bilayer to effectively deliver a protein into cells. Also, it is known that, when HA2 and PTD are used at the same time, the secretion of heterologous molecules (such as polypeptides or proteins) from the endosome into the cytoplasm, the nucleus or other intracellular organs will be increased to promote the permeation of the molecules into cells (US 2006/0222657A1).

Peptides known to have a function similar to that of HA2 include an influenza virus-derived diINF-7 domain, a hepatitis B virus (HBV)-derived TLM

(translocation motif), a human papilloma virus (HPV)-derived L2 domain, a

Histatin 5 domain developed as an antibiotic, and dhvar4 and dhvar5 domains as synthetic peptides (Stoeckl, L. et al., Proc. Natl. Acad. ScL, 103:6730 2006;

Kamper, N., J. Virol., 80:759, 2006; Mastrobattista, E. et al, J. Biol. Chem., 277:27135, 2002; den Hertog, A.L. et al., Biochem. J., 379:665, 2004). These peptides allow the virus or peptide delivered into cells to be released from the lipid membrane to the cytoplasm (endosomal escape or endosomal rescue).

In the present invention, hAxin2 of SEQ ID NO: 1, which is an Axin2-derived peptide, is used as the GBD, but the scope of the present invention is not limited thereto. For example, other animal-derived peptides, such as SEQ ID NO: 2 to

SEQ ID NO: 7, may be used, and any peptide may also be used, as long as it can bind to GSK3 to inhibit the nuclear export of GSK3. The peptides of SEQ ID

NO: 1 to SEQ ID NO: 7 bind to GSK3 competitively with Axin so as to inhibit the nuclear export of GSK3.

In addition, when a nuclear localization signal (NLS) is included in the inventive polypeptide during the synthesis of the peptide, the polypeptide can more effectively inhibit the GSK3 nuclear export function of Axin. Typical examples of NLS include PKKKRKV (SEQ ID NO: 8) derived from the large T- antigen of Simian Virus-40, but it is possible to use NLS and combinations thereof, which are present in various viruses and many proteins. When this NLS (nuclear localization signal) is included in the polypeptide, the pharmacological effect of the polypeptide can be improved. The effect of increasing the concentration of GSK3 in the nucleus according to the present invention can be amplified through the use of NLS. For example, as shown in FIG. 27, it can be seen that NLS allows the polypeptide to be imported into the nucleus and amplifies the effect of the polypeptide.

In another aspect, the present invention relates to a cell-permeable polypeptide for inhibiting the nuclear export of GSK3 and to a compound having a function similar thereto, which comprise: a PTD (protein transduction domain), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; an Axin-binding domain (ABD), which binds to Axin to inhibit the nuclear export (from nucleus to cytoplasm) of GSK3; a HA2 domain (HD) capable of avoiding a decrease in biological activity, caused by macropinosome or endosome; and a nuclear localization signal, which enables the polypeptide to be easily imported into the nucleus so as to effectively inhibit the GSK3 nuclear export function of Axin.

In the present invention, a GSK3-derived peptide cloned using primer of SEQ ID NO: 9 and 10 is used as the ABD, but the scope of the present invention is not limited thereto.

SEQ ID NO: 9 (GFP-ABL-F): 5'- atg gac gag ctg tac aag ggt acctta eta gga caa cca ata SEQ ID NO: 10 (GFP-ABL-R): 5'- tat tgg ttg tec tag taa ggt ace ctt gta cag etc gtc cat

For example, any peptide may be used as the ABD without any particular limitation, as long as it can bind to Axin2 so as to restrict the GSK3 nuclear export function of Axin. The peptide cloned using the primer of SEQ ID NO: 8 or SEQ ID NO: 9 binds to Axin competitively with GSK3 so as to inhibit the GSK3 nuclear export function of Axin.

Wnt signaling leads to a decrease in the expression of E-cadherin and an increase in the expression of β-catenin and Snail and causes the invasive growth and metastasis of cancer cells (Yook, J.I. et al., J. Biol. Chem., 280:11740, 2005). The regulation of phosphorylation of Snail by /3-catenin is schematically shown in FIG. 2. In brief, /3-catenin increases the expression of Axin, and Axin binds to GSK3 in the nucleus so as to export GSK3 to the cytoplasm. For this reason, the expression of Snail is increased, and the metastasis of cancer cells occurs. The process of regulating the concentration of GSK3 in the nucleus by Wnt signaling is schematically shown in FIG. 3. Thus, when a polypeptide or compound having a structure, which is the same as or similar to the GSK3- binding domain of Axin, is used, the GSK2 nuclear export function of Axin can be inhibited, and thus the development and progression of various diseases, caused by Wnt signaling and /3-catenin activation, can be inhibited. FIG. 4 shows an Axin-GSK3 binding structure, and from FIG. 4, it can be seen that Axin binds to GSK3 by an Axin-derived peptide fragment (the sequence of amino acids 370-390 of hAxin2 (Dajani et al, EMBO J., 22:494, 2003). Herein, the Axin and FRAT proteins have an α-helix structure, and the hydrophobic residues in the middle thereof bind to the groove structure of GSK3. Thus, a polypeptide, which can bind to Axin competitively with GSK3, can inhibit the GSK3 nuclear export function of Axin or FRAT. Also, the FRAT genes bind to GSK3 using a structure very similar to Axin, and thus polypeptides derived from the FRAT genes, or polypeptides having α-helix and hydrophobic residues similar to those of the FRAT genes, can inhibit the concentration of GSK3 in the nucleus by inhibiting the nuclear export of GSK3.

The present invention also suggests the new function and regulatory mechanism of /3-catenin, which has been known to be the most important factor in cancer developmental processes (Giles, R.H. et al., Biochim. Biophys. Acta, 1653: 1, 2003). Accordingly, the present invention suggests that /3-catenin functions to induce not only cancer development, but also the invasive growth and metastasis of cancer cells. Generally, because genetic mutations or other abnormalities in the Wnt signaling system are observed in more than 90% of cancers occurring in humans, it is thought that the Wnt signaling system is most important in cancer development, but a concrete regulatory mechanism for the Wnt signaling system is not well known. It is merely known that the genetic abnormalities in the Wnt signaling system commonly activate the /3-catenin gene, thus causing a cancer developmental process (Reya, T. & Clevers, H., Nature, 434:843, 2005; Giles, R.H. et al, Biochim. Biophys. Acta, 1653: 1 , 2003). However, the concept and cytological mechanism, in which the activation of the /3-catenin gene induces the invasive growth or metastasis of cancer cells, are not known yet.

In the present invention, the activation of the /3-catenin gene induces the expression of the Axin2 gene, and the Axin2 gene binds to GSK3 in the nucleus so as to perform the nuclear export of GSK3, thus reducing the concentration of

GSK3 in the nucleus. For this reason, the expression of the Snail gene, which is phosphorylated and degraded by GSK3, is increased. As a result, an increase in the expression of the Snail gene, caused by Wnt signaling, induces the invasive growth and metastasis of cancer cells, and clinically the continuous recurrence and distant metastasis of cancers (Yook, J.I. et al., J. Biol. Chem., 280: 11740,

2005).

Up to date, most anticancer agents or /3-catenin targeting therapeutic agents have been developed as materials which induce the apoptosis of cancer cells.

Therefore, the signaling mechanism suggested in the present invention suggests a new therapeutic effect and concept of /3-catenin targeting agents, which have been developed to date or will be developed in the future. As methods for inhibiting the activity of /3-catenin, methods of inhibiting the transcriptional regulation by (3-catenin through competitive binding to /3-catenin transcriptional complexes, such as LEF/TCF, CBP300 and the like, are mainly known (Poy, F. et al, Nature Structural Biol,, 8: 1053, 2001 ; Clevers, H., Nature Rev. Drug Discov.,

5:997, 2006). In this case, most of drugs developed to date have been developed for the effect of inducing the apoptosis of cells by targeting /3-catenin. However, the present invention suggests the possibility that these drugs ultimately inhibit the expression of Axin2 to increase the expression of GSK3 in the nucleus and to inhibit the metastasis or recurrence of cancer cells. Particularly, in the treatment of cancer patients, if the prior 0-catenin targeting therapeutic agent or the complex suggested in the present invention is used to inhibit or regulate the continuous recurrence and distant metastasis of cancer cells, the survival rate and survival time of the cancer patients can be increased by at least two times, and furthermore, the cancer treatment concept, which has been maintained for 30 years or more, can be significantly changed.

In another aspect, the present invention relates to a pharmaceutical composition for inhibiting the growth and metastasis of cancer cells or a pharmaceutical composition for treating immune diseases, the compositions containing, as an active ingredient, a polypeptide for inhibiting export of GSK3 from nuclear to cytoplasm.

The polypeptide according to the present invention will be useful for inhibiting not only the metastasis of cancer cells, which is stimulated by Wnt signal transduction, but also the development and progression of autoimmune degenerative diseases such as rheumatic arthritis.

The polypeptide according to the present invention can be used by itself or in the form of pharmaceutically acceptable acid addition salts or metal complexes, for example, of zinc or iron salts. More specifically, the acid addition salt is preferably selected from the group consisting of hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate and tartrate.

The pharmaceutical composition containing the inventive polypeptide as an active ingredient is preferably prepared by mixing and diluting the active ingredient with a pharmaceutically acceptable excipient or matrix carrier or by sealing the active ingredient into a receptacle-like carrier, depending on administration routes and modes and intended therapeutic uses. Moreover, the inventive composition may also be used in combination with other drugs useful for the treatment of bone defects. In this case, the preparation of a physiologically acceptable composition having the desired pH, isotonicity and stability may be performed using any conventional method known in the art to which the present invention pertains. The matrix used in the present invention may be selected depending on bioadhesion, biodegradability, mechanical properties, attractive appearance and contact properties. Examples of the carriers, which can be used in the present invention, include biodegradable and chemical substances, such as calcium sulfate, tricalcium phosphate, hydroxyapatite and polylactic acid; biodegradable and biological substances, such as bone or skin collagens, and other pure proteins or cellular matrix components; non-biodegradable and chemical substances, such as sintered hydroxyapatite, bioglass, aluminate and other ceramics; combinations of the above substances, such as polylactic acid, hydroxyapatite, collagen and tricalcium phosphate. However, the present invention is not limited to the above-mentioned carriers.

Examples of excipients, which can be used in the present invention, include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, and mineral oil.

Meanwhile, the pharmaceutical composition containing the inventive polypeptide is preferably used in the form of an injection solution or capsule in order for the composition to be introduced into therapeutic sites. The dose of the inventive composition can be determined considering the kind of excipients or matrix carriers used, the therapeutic sites of patients, the patient's age, sex and diet, disease severity, administration period, and other clinical factors. Thus, for example, a conventionally known effective amount of the composition may be administered at once or at intervals, considering the patient's weight, and additional administration can be determined while the therapeutic effect is observed.

Although the polypeptide according to the present invention can be conveniently constructed by synthesis, it may also be prepared by culturing E. coli or other bacteria, yeasts or fungi, transformed with a recombinant vector comprising a base sequence encoding each of the domains of the polypeptide. Particularly, the polypeptide according to the present invention can be prepared in a large amount in a form fused with a carrier protein using recombinant bacteria, and the prepared polypeptide may have an effect similar to that of the synthesized polypeptide.

Accordingly, in still another aspect, the present invention relates to a recombinant vector for preparing a cell-permeable polypeptide for inhibiting the development and progression of diseases, which occur due to a decrease in the concentration of GSK3, recombinant microorganisms transformed with said recombinant vector, and a method for preparing a polypeptide for inhibiting the nuclear export of GSK3, the method comprising culturing said recombinant microorganisms.

In the present invention, as shown in FIG. IB, a tag for confirming the isolation, purification and expression of the polypeptide, a base sequence encoding the GSK3 binding domain for inhibiting hAxin2-GSK3 binding, a base sequence encoding the nuclear localization signal (NLS), a base sequence encoding HA2 for macropinosome rescue, and a base sequence encoding the cell-permeable PTD (protein transduction domain), were inserted into a pRSET vector, thus constructing a recombinant vector. Then, recombinant microorganisms transformed with said recombinant vector were cultured, thus preparing a polypeptide for inhibiting the development and progression of diseases, which occur due to a decrease in the concentration of GSK3.

As used herein, the term "vector" refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. The examples of the vector include plasmids, phage particles, or simply potential genomic inserts. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. As used herein, the term "plasmid" and "vector" are sometimes used interchangeably, because the plasmid is the most commonly used form of vector at present. For the purpose of the present invention, the plasmid vector is preferably used. A typical plasmid vector which can be used for this purpose contains the following: (a) a replication origin by which replication occurs efficiently such that several hundred plasmid vectors per host cell are created; (b) an antibiotic-resistant gene by which host cells transformed with the plasmid vector can be selected; and (c) restriction enzyme digestion sites into which foreign DNA fragments can be inserted. Even if suitable restriction enzyme digestion sites are not present in the vector, the use of a conventional synthetic oligonucleotide adaptor or linker enables the easy ligation between the vector and the foreign DNA fragments.

After ligation, the vector should be transformed into suitable host cells. The transformation can be easily achieved using the well-known calcium chloride method or electroporation (Neumann et ai, EMBO J., 1 :841 , 1982).

For the overexpression of a gene, an expression vector known in the art may be used in the present invention.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and present in open reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As well-known to the art, in order to increase expression level of a transfected gene in a host cell, the gene should be operably linked to transcriptional and translational control sequences which can work in a selected expression host. Preferably, the corresponding gene and expression control sequence are contained in an expression vector comprising a selection marker and replication origin. When an expression host is a eukaryotic cell, the expression vector should further comprise an expression marker useful in the eukaryotic expression host cell.

The host cell transformed with said recombinant vector constitutes another aspect of the present invention. As used herein, the term "transformation" refers to introducing DNA into a host cell so that the DNA is replicable, either as a chromosomal integrant or as an extrachromosomal element.

Of course, it should be understood that all the vectors do not function equally to express the DNA sequences according to the present invention. Likewise, all the host cells do not function equally for the same expression system. However, those skilled in the art may properly select a vector, expression control sequence and host cell without departing from the scope of the present invention and without undue experimentation. For example, in selection of a vector, a host cell must be considered. This is because the vector should be replicated therein. Also, the replication number and ability to control the replication number of a vector and expression of other proteins encoded by the vector, for example, antibiotic marker, should be considered.

The polypeptide according to the present invention may be used not only as a therapeutic agent, but also in drug screening and development, intracellular genetic manipulation, imaging for patient diagnosis, and the like, after it is bound to various nanosized materials or quantum dots (Q-dots), which have been recently broadly developed (Kandere-Grzybowska, K. et al, Nature Methods, 2:739, 2005; Gao, X. et al, Nature Biotechnol, 22:969, 2004; Xie, X.S. et al, Science, 312:228, 2006; Rosi, N.M. et al, Science, 312: 1027, 2006; Giepmans, B.N.G. et al, Science, 312:217, 2006; Won, J. et al, Science, 309: 121 , 2005). For example, when live cells are labeled with GSK3 and the inventive polypeptide by Q-dots having different emission wavelengths and when the binding of two kinds of Q-dots treated with various concentrations of various compounds is observed in live cell imaging, the efficiency, specificity and reliability of this method can be greatly increased compared to those of the prior chemical screening method.

When the inventive polypeptide bound to nanosized materials or Q-dots is administered into cells, it can inhibit the invasive growth or metastasis of cancer cells. Thus, in another aspect, the present invention relates to a formulation for treating diseases caused by a decrease in the concentration of GSK3 in the nucleus, the formulation comprising the inventive polypeptide bound to nanosized materials or quantum dots (Q-dots).

In still another aspect, the present invention relates to a gene therapy agent, comprising: a therapeutic gene encoding the inventive polypeptide; and a carrier thereof. In the present invention, as the carrier, a viral vector, such as retrovirus or baculovirus, which are generally used as gene carriers, may be used, but the scope of the present invention is not limited thereto.

Also, when the inventive polypeptide bound to nano-sized materials or quantum dots (Q-dots) is administered into cells, cell kinetics can be traced by real-time cell imaging, and thus cancer cells or the like can be distinguished from normal cells. Accordingly, in still another aspect, the present invention relates to a diagnostic agent for diagnosing diseases caused by a decrease in the concentration of GSK3 in the nucleus, the diagnostic agent comprising the inventive polypeptide bound to nanosized materials or quantum dots (Q-dots).

According to the present invention, the mechanism of the GSK3 nuclear export function (function of exporting GSK3α and GSK3/3 from the nucleus to the cytoplasm) of Axin in the Wnt signaling pathway was clearly established, and thus the method for substantially inhibiting the decrease in the concentration or activity of GSK3 in the nucleus or maintaining or increasing the concentration or activity thereof was suggested for the first time. That is, Wnt signaling increases the transcription and activity of Axin2 through β-catenin, and thus reduces the concentration of GSK3 in the nucleus through the GSK3 nuclear export function of Axin2. The activation of j8-catenin by Wnt signaling leads to the activation of Axin2 having the GSK3 nuclear export function so as to reduce the concentration of GSK3 in the nucleus, thus inhibiting the phosphorylation of various regulatory factors in the nucleus.

Accordingly, in still another aspect, the present invention relates to a method for inhibiting a decrease in the concentration of GSK3 in the nucleus, which is caused by caused by the nuclear export of GSK3 through binding to Axin, the method comprising: (a) blocking the Axin-binding domain of GSK; (b) blocking the GSK3-binding domain of Axin; (c) inhibiting the activation of Axin; and (d) increasing the expression of GSK3 in the nucleus.

In the present invention, blocking the Axin binding domain of GSK3 can be performed using a compound, a peptide or a polypeptide, which bind to the Axin binding domain of GSK3. Also, blocking the GSK3 binding domain of Axin can be performed using a compound, a peptide or a polypeptide, which bind to the GSK3 binding domain of Axin. The polypeptide is preferably the cell- permeable polypeptide according to the present invention.

Inhibiting the activation of Axin can be performed by inhibiting Wnt signaling in the Wnt/j8-catenin pathway or by inhibiting the activity of /3-catenin. In addition, the formulation and gene therapy agent according to the present invention may also be used to inhibit a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin.

Examples

Hereinafter, the present invention will be described in further detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are illustrative only, and the scope of the present invention is not limited thereto.

Example 1 : E-cadherin expression and /3-catenin activation by Wnt signaling

MCF-7 cells (ATCC, American Type Culture Collection, HTB-22) labeled with fluorescent nanobeads, into which a Mock expression vecto^MCF-T/Mock) have been introduced, and MCF-7 cells, into which an Wnt-1 expression vector (MCF- 7/Wnt) have been introduced, were fixed in 4% formaldehyde and added to 0.1% Triton- 100. Then, the cells were stained with an anti-E-cadherin antibody (Zymed Laboratories) and an anti-/3-catenin antibody (BD Bioscience), and then stained with an Alexa-Fluor-594-labeled secondary antibody. Then, the stained cells were observed under an electron microscope. Torto-3 (molecular probe) or DAPI (molecular Probe) was used to stain the nucleus. As a result, it could be seen that, in the MCF-7/Wnt cells, the expression of E-cadherin was reduced, and /3-catenin was activated (FIG. 5).

Example 2: Activation and metastasis of E-cadherin by Wnt signaling and Snail

The expressions of β-catenin and Snail in the MCF-7/Mock cells and the MCF- 7/Wnt cells were examined by Western blot analysis. As a result, in the MCF- 7/Wnt cells, the expression of E-cadherin was reduced, but the expression of β- catenin and Snail were increased (FIG. 6A). This suggests that the expression of E-cadherin is reduced due to Wnt signaling.

In the MCF-7/Mock cells and the MCF-7/Wnt cells, the Snail gene was knocked down with short hairpin RNA (sh-RNA) (Snail-sh-RNA), and then the activation of the E-cadherin promoter was examined. As a result, the activity of the E- cadherin promoter was increased (FIG. 6B). This suggests that the activity of E- cadherin is reduced due to the Snail gene.

MCF-7 cells were labeled with fluoresbrite carboxylate nanospheres (Polysciences, Inc.) and cultured on the chick chorioallantoic membrane (CAM) of 1 1 -week-old chick embryos. Then, the invasion of the cells on the cut surface of the fixed CAM was observed with a fluorescence microscope. As a result, the MCF-7/Mock cells remained on the CAM surface and did not show metastasis, but the MCF-7/Wnt cells showed metastasis across the CAM surface. Also, in the case of the MCF-7/Wnt cells, E-cadherin was expressed at the cell- cell boundary, and /3-catenin was imported into the nucleus (FIG. 6C). This suggests that Wnt signaling inhibits the metastasis of cancer cells through the stabilization of the Snail gene.

In the case where the Snail gene in the MCF-7/Wnt cells was knocked-down with short hairpin RNA (Snail-sh-RNA), metastasis did not occur (FIG. 6D). This suggests that the Snail gene is involved in metastasis.

Example 3: Activation and metastasis of E-cadherin by /3-catenin

The activation of the E-cadherin promoter in transformed cells (S33 Y-β-catenin), obtained by introducing an S33Y /3-catenin expression vector (Yook et ah, Nature Cell Biol. , 8: 1398, 2006) into MCF-7 cells, and in the MCF-7/Mock cells, was examined. As a result, in the S33Y-/3-catenin cells, the cell metastasis occurred, and the activation of the E-cadherin was reduced, similar to the MCF- 7/Wnt cells (FIG. 7A). This suggests that the invasive growth and metastasis of cancer cells can be stimulated only by the activation of /3-catenin. In addition, it shows that _/3-catenin targeting drugs are effective not only in the apoptosis of cancer cells, but also in inhibiting the metastasis of cancer cells.

Also, a dominant-negative TCF (DN-TCF) expression vector was introduced into the MCF-7/Wnt cells and cultured on CAM (chick chorioallantoic membrane). Then, the metastasis of the cells was examined and, as a result, the metastasis did not occur (FIG. 7B).

Example 4: Snail gene expression and E-cadherin regulation by /3-catenin

The Topflash activities of Mock, Snail and S33Y were measured and, as a result, S33Y showed the highest activity (FIG. 8A). This suggests that the activation of β-catenin increases the half-life of the Snail gene, thus increasing the expression of the gene.

The expression of Snail mRNA in the transformed cells, obtained by introducing the S33Y /5-catenin expression vector into the MCF-7 cells, was analyzed by RT- PCR. As a result, the MCF-7/Mock cells and the MCF-7/S33Y cells showed similar levels of expression of Snail mRNA. This suggests that the expression of Snail mRNA is not induced by β-catenin (FIG. 8B).

MCF-7 cells were co-transfected with an S33Y β-catenin-myc expression vector, a Snail-flag expression vector and/or a DN-TCF-Flag expression vector, and the expression of the Snail gene in the cells was examined. As a result, in the transformed cells, obtained by introducing the S33Y /?-catenin-myc expression vector and the Snail-flag expression vector into MCF-7 cells, the expression of the Snail gene was increased, whereas, in the transformed cells, obtained by co- transfecting the MCF-7 cells with the S33Y /3-catenin-myc expression vector, the Snail-flag expression vector and the DN-TCF-Flag expression vector, the expression of Snail was reduced (FIG. 8C). This suggests that the /3-catenin- TCF flag regulates the expression of the Snail gene.

The expression of Snail mRNA in the transformed cells (MCF-7/S33Y), comprising the S33Y β-catenin expression vector introduced into MCF-7 cells, was examined. As a result, the expression of Snail in the MCF-7/S33Y cells was increased (FIG. 8D). This suggests that /3-catenin increases the expression of Snail.

The activities of E-cadherin in the MCF-7/Mock cells, the MCF-7/S33Y cells, the transformed cells (MCF-7/S33Y DN-TCF), obtained by co-transfecting S33Y β- catenin and epitope-tagged Snail into MCF-7 cells, the transformed cells (MCF- 7/S33Y Scr), obtained by introducing S33Y /?-catenin and Scr into MCF-7 cells, and the transformed cells (MCF-7/S33Y Snail siRNA), obtained by introducing Snail gene having knocked down of S33 Y β-catenin and a small inhibitory RNA (siRNA) into MCF-7 cells, were measured.

As a result, in the MCF-7/S33Y cells and the MCF-7/S33Y Scr cells, the activity of E-cadherin was reduced, but in the MCF-7/S33Y DN-TCF cells and the MCF- 7/S33Y Snail siRNA cells, the activity of E-cadherin was increased (FIG. 9A). This suggests that the activation of _/3-catenin reduces the expression of E- cadherin through the Snail gene. Also, as shown in FIG. 9B, the MCF-7/S33Y Scr cells showed metastasis, whereas the MCF-7/S33Y Snail siRNA cells did not show metastasis.

Example 5: Expression and metastasis of Snail by Axin2

In order to examine the effect of the activation of /3-catenin on the expression of Axin2, the expressions of Axinl and Axin2 mRNA in the MCF-7/S33Y cells were measured by RT-PCR. As a result, the expression of Axinl in the MCF- 7/S33 Y cells was not changed, but the expression of Axin2 was increased (FIG. 10A). This indicates that /3-catenin increases the expression of Axin2. Also, whether Snail was expressed in the presence of Axin2 was examined. As a result, in the case of the MCF-7/Mock cells, Snail and Axin2 were not expressed, but in the case where the Axin2 expression vector was introduced into MCF-7 cells, the expression of Snail was increased (FIG. 10B).

The half-life of Snail in the presence of Axin2 was measured by pulse-chase analysis. MCF-7 cells were co-transfected with 1.0 μg of a pCR3.1-Snail- FLAG expression vector and 1.0 μg of a mock expression vector or 1.0 μg of an Axin2 expression vector, and then labeled with 50μ Ci/m£ [³⁵S]Met/Cys (PerkinElmer Life Sciences) in Met/Cys-free medium for 20 minutes. The labeled cells were washed and cultured for 0, 2, 4 and 8 hours. The cell lysate was subjected to immunoprecipitation with anti-FLAG-M2-agarose beads, followed by performing SDS-PAGE/autoradiography. As a result, in the MCF- 7/Snail/Mock cells, the half-life of Snail was reduced with the passage of time, but in the MCF-7/Snail/Axin2 cells, the half-life of Snail was maintained without changes. This suggests that Axin2 increases the half-life of Snail (FIG. 10C).

MCF-7 cells were co-transfected with Axin2 and Scr, or co-transfected with Axin2 and Snail siRNA (small inhibitory RNA), and then cultured on the CAM surface. Then, the metastasis in the cultured cells was analyzed. As a result, as shown in FIG. 10D, in the MCF-7/ Axin2/Scr cells, the metastasis occurred, whereas, in the MCF-7/Axin2/Snail siRNA cells, the metastasis did not occur. This suggests that the activation of /3-catenin increases the expression of Axin2, Axin2 increases the half-life of Snail so as to be stabilized, and stimulates the invasive growth and metastasis of Snail. That is, a process of inhibiting the phosphorylation of Snail by /3-catenin occurs due to Axin2.

Example 6: Export of GSK3/3 by Axin2

In order to examine the export of GSK3_/8 by Axin2, HA-tagged GSK3/3 and mock expression vector was introduced into MCF-7 cells or HA-tagged GSK30 and Flag-tagged Axin2 were introduced into MCF-7 cells, and then the cells were stained with an anti-HA antibody for examining the GSK3/3 location, an anti-Flag antibody for examining the Axin2 location, or Toto-3 for examining the nucleus. The stained cells were observed by confocal laser microscopy. As a result, it could be observed that GSK3/3 was located in the cytoplasm and nucleus in the transformed cells into which the mock expression vector was introduced, but was located in the cytoplasm in the transformed cells into which Axin2 was introduced (FIG. HA). Also, the expression of GSK3/3 in the total cell lysate and nuclear extract of the transformed cells was examined. As a result, in the total cell lysate, the expression level of GSK3/3 was maintained, but in the transformed cells into which Axin2 was introduced, the expression of GSK3 in the nuclear extract was reduced, and the expression of Snail was increased (FIG. HB). This suggests that Axin2 exports GSK3 from the nucleus, so that the Snail phosphorylation by GSK3 is inhibited.

MCF-7 cells were co-transfected with wild-type, D9-GSK3/3 and Y216F-GSK3/3, and the Snail expression vector, and then the expressions of GSK3/5 in the total cell lysate and the nuclear extract were examined. As a result, the wild-type,

D9-GSK3/3 (Δ9) and Y216F-GSK3/3 were expressed in the total cell lysate at the same level, but the expression of Y216F-GSK3/3 in the nuclear extract was reduced. Also, in the nuclear extract, the Snail expressions of wild-type and D9-GSK3_/3 were reduced (FIG. HC). This indicates that the concentration of

GSK3 in the nucleus regulates the expression of Snail.

Example 7: Action mechanism of Axin2 on GSK3

FIG. 12A shows a leucine-rich nuclear export signal (NES) which regulates nuclear trafficking via the nuclear receptor-dependent pathway of chromosome maintenance region 1 (CRMl) present in human, mouse and chicken Axin2 genes. NES-I , NES-2 and NES-3 result from leucine-to-arginine mutation.

The transformed cells into which Axin2 was introduced were cultured in the presence of leptomycin B (LMB) as a CRMl inhibitor, and the locations of Axin2 and GSK3/3 (glycogen synthase kinase-3/3) were examined. As a result, when the cells were treated with LMB, Axin2 and GSK3/3 were located in the nucleus, and the expression thereof in the nuclear extract was increased (FIG. 12B). This suggests that the nuclear export signal (NES) present in Axin2 plays a role in export of GSK3 from nucleus to cytoplasm.

Also, a mock expression vector or introduced with Snail expression vector-bound wild-type Axin2, NES-I , NES-2, NES-3 and GSK3-binding domain-deleted (ΔGSK3) expression vector were introduced into MCF-7 cells. Then, the cells were analyzed for the expression of GSK3/3 and the activity of the E-cadherin promoter. As a result, in the case of the wild- type Axin2, NES-I, NES-2 and NES-3 transformants, the expression of GSK3/3 in the total cell extract was maintained, but in the case of the wild-type Axin2, NES-I and NES-2, the expression of GSK3/3 in the nucleus was reduced. Also, in the case of the NES- 3 and ΔGSK3 transformants, the expression of GSK3/3 in the nucleus was maintained at a normal level, and the expression of Snail was reduced. With respect to the activity of the E-cadherin promoter, it was reduced in the wild-type Axin2, NES-I, NES-2 and NES-3 transformants (FIG. 12C). This indicates that Axin2 exports GSK3 from the nucleus to the cytoplasm and increases the expression of Snail, thus reducing the expression of E-cadherin.

Example 8: Induction of Axin2 gene expression and regulation of GSK3 and Snail in nucleus by /3-catenin

MCF-7 cells were co-transfected with S33 Y /3-catenin and Snail expression vectors, and then the expression of Axin2 mRNA was analyzed by RT-PCR, and the amounts of GSK3 and Snail in the nucleus were analyzed by Western blot.

As a result, due to S33Y _/3-catenin, Axin2 mRNA was increased, the amount of GSK3 in the nucleus was reduced, and the amount of Snail in the nucleus was increased (FIG. 13). This suggests that the activation of _/3-catenin increases the expression of Axin2, the concentration of GSK3 in the nucleus is reduced due to the GSK3 nuclear export function of Axin2, and thus the invasion and metastasis of cancer cells occur due to the stabilization of Snail and the reduction in the expression of E-cadherin.

Example 9: Effect of Axin2 on Snail expression in human cancer tissue

The expressions of Axin2 and Snail in human normal breast tissue and breast cancer tissue were observed by immunohistochemistry. Breast cancer tissue and normal breast tissue in paraffin, obtained from Pathology room, Ilsan Hospital in Korea, were deparaffined, and then hydrated with xylene and graded alcohol. Then, antigens were detected from the tissues using a citrate buffer (DakoCytomation, Denmark) and microwave heating, and the samples were stained with 3-amino-9-ethyl-carbazole and methyl green using an automatic system by an avidin-biotin complex peroxidase technique (Techmate 500+, DakoCytomation). As a result, the expressions of Axin2 and Snail were increased depending on the progression of cancer (FIG. 14).

The expressions of Axin2 mRNA and Snail in the breast cancer tissues, in which venous invasion and lymph node metastasis was detected, were observed. As a result, it could be observed that the expressions of Axin2 and Snail were all increased (FIG. 15). This suggests that the increase in Snail expression by Axin2 expression is important not only in cytological terms, but also in actual human breast cancer.

Also, human cervical cancer cells were co-transfected with Axin2 and GSK3, and the expressions of Axin2 and GSK3 in the cytoplasm and the nucleus were analyzed. As a result, the induction of Axin2 expression in human cervical cancer cells led to a decrease in the concentration of GSK3 in the nucleus and to an increase in the expression of Snail (FIG. 16).

The expressions of Wntl mRNA and Wnt3a mRNA in human cervical tissue were observed by immune staining. As a result, the expressions of Wntl mRNA and Wnt3a mRNA were increased with the progression of cancer (FIGs. 17 and 18).

The expressions of Axin2 mRNA in cervical precancerous lesions and cancer cells were examined and, as a result, the expression of Axin2 mRNA was increased (FIG. 19). Also, during the development of cervical cancer, the expression of Snail was increased (FIG. 20).

shSnail (small hairpin RNA interference Snail) and shAxin2 (small hairpin RNA interference Axin2) were introduced into a invasive-metastatic cervical cancer line. When the Snail and Axin2 gene in the transformed cells were knocked-out, the invasive growth and metastasis of the cancer cells were completely blocked (FIG. 21).

Also, Wntl or Wnt3a in the cells was expressed and, as a result, the expressions of GSK3α and GSK3/3 in the nucleus were all reduced (FIG. 22). This suggests that Wnt signaling leads to a decrease in the expression of GSK3 in the nucleus.

Example 10: Construction and application of polypeptide and expression vector, which increase expression of GSK3 in nucleus through specific binding to GSK3

In order to specifically and effectively inhibit the GSK3 nuclear export function of Axin, an Axin2 domain capable of binding to GSK3 was used to construct a GFP-fusion (green fluorescent protein-fusion) expression vector (FIG. 23). Particularly, in order to induce binding to GSK3 in the nucleus, a vector comprising three SV40 large T-antigen-derived nuclear localization signals (NLS: PKKRKV: SEQ ID NO: 8) linked therewith was also separately constructed using PCR (polymerase chain reaction). Herein, as GFP, a pCMS- EGFP expression vector (Clontech, USA) was used, and the GFP-NLS fusion vector was constructed by PCR using primers of SEQ ID NO: 1 1 and SEQ ID NO: 12. That is, GFP was cloned from pCMS-GFP, and three continuous NLSs at the C-terminal end of GFP were constructed by PCR amplification and cloned into the Hindlll-Xbal sites of human cell expression vector pcDNA4-myc-his (Invitrogen, USA). In addition to this vector, GFP fusion vectors or vectors for TAT-recombinant fusion proteins, which will be described later, were all amplified by multi-step PCR.

SEQ ID NO: 11 : 5'-ctttttcttaggcaccgccacctttctcttctttttcggggtacccttgtacagctcgtccat

SEQ ID NO: 12: 5'-ggctccacctctagatgggaccttccgtttcttctttggggcgggaactttacgctttttcttaggcac cgc

In order to fuse the GSK3 binding domain of Axin to GFP or the GFP-NLS expression vector, PCR was performed using primers of SEQ ID NO: 13 to SEQ ID NO: 16 in the same manner as described above. Specifically, each of the GFP expression vector and the GSK3 -binding domains of the Axin2 expression vector was primarily amplified with said primers, and then the vectors were secondarily amplified together, to construct the fusion protein vector, thus being cloned into the same sites of the vector described above. GFP-F (SEQ ID NO: 13): 5'-gga ggc eta ggc ttt tgc

GFP-Axin-F (SEQ ID NO: 14): 5'-atg gac gag ctg tac aag ggt ace gag atg ace ccc gtg gaa GFP-Axin-R (SEQ ID NO: 15): 5'-ttc cac ggg ggt cat etc ggt ace ctt gta cag etc gtc cat GFP-R (SEQ ID NO: 16): 5'-cct gcc gcc tct aga gcg get etc caa etc cag

Also, in order to examine the role of hydrophobic residues at the α-helix site, the residues were substituted with hydrophilic residue arginine (Arg), thus constructing a control group. For this the following primers were used. GBD-arg-Rl (SEQ ID NO: 17): 5'-cttttcccgcctcgagatccgctcagctgcacgggtggcgggttccac

GBD-arg-R2 (SEQ ID NO: 18): 5'-cctgccgcctctagagcggctctcccgctcccgcttccgcttttcccgcctcgagat

In addition, in order to identify the role of a GSK3 phosphorylation site (present in the front of the GSK3 binding domain) in the GSK3 nuclear export function of Axin and to apply the GSK3 phosphorylation site, an expression vector comprising the GSK3 phosphorylation site was constructed. As a control group, a vector, in which the phosphorylation site was substituted with alanine (Ala), was constructed. That is, serines at locations 296, 300 and 304 of Axin2 were substituted with alanine, primers containing said locations were constructed, and then PCR amplification was performed. Herein, the following primers were used.

NLS-GBD-F (SEQ ID NO: 19): 5'-aaa egg aag gtc cca tct gag atg ace ccc gtg gaa NLS-GBD-R (SEQ ID NO: 20): 5'-ttc cac ggg ggt cat etc aga tgg gac ctt ccg ttt NLS-GBD-GP-F (SEQ ID NO: 21): 5'-aaa egg aag gtc cca tctaat cct tat cac ata ggt NLS-GBD-GP-R (SEQ ID NO: 22): 5'-acc tat gtg ata agg att aga tgg gac ctt ccg ttt

Each of the vectors was transfected into the 293 cell line (ATCC, CRL- 1573) using Lifectamine 2000 (Invitrogen, USA), the effect thereof on an increase in the concentration of GSK3 in the nuclear fraction and the specificity thereof were examined according to the above-described method. Among these vectors, the domain proved to have the effects of reducing the expression of Snail and inhibiting the invasive growth of cancer cells is useful for the construction of a polypeptide for inhibiting the export of GSK3 or is useful for the construction of an expression vector for gene therapy. When the expression vector or its fragment is inserted into a conventional gene carrier such as retrovirus or when the polypeptide bound to nano-sized materials or quantum dots (Q-dots) is administered into cells, it can be used as a gene therapy agent for treating diseases, which occur due to a decrease in the concentration or expression of GSK3 in cells.

Example 11 : Construction of polypeptide for inhibiting GSK3-Axin binding

The GSK3 binding domain of Axin2 was used to construct a cell-permeable polypeptide for inhibiting the GSK3 nuclear export function of Axin2 and increasing the concentration of GSK3 in the nucleus. That is, a tag for confirming the isolation, purification and expression of the polypeptide, a base sequence encoding the GSK3 binding domain (SEQ ID NO: 1 ; amino acids 370- 390 of hAxin2) for inhibiting hAxin2-GSK3 binding, a base sequence encoding the nuclear localization signal (NLS) for nuclear import during the synthesis of the peptide, a base sequence encoding HA2 for macropinosome rescue, and a base sequence encoding the cell-permeable PTD (protein transduction domain), were inserted into a pRSET vector, thus constructing a recombinant vector (FIG. IB). Then, recombinant microorganisms transformed with said recombinant vector were cultured, thus preparing a polypeptide for inhibiting the development and progression of diseases, which occur due to a decrease in the concentration of GSK3.

Example 12: Construction and application of GSK3 domain-derived Axin binding polypeptide and expression vector

When a GSK3-derived polypeptide is used in the GSK3-binding domain of Axin, the GSK3 nuclear export function of Axin can be effectively inhibited. In order to verify it, an expression vector containing a base sequence encoding a GSK3- derived peptide capable of binding to Axin was constructed (FIG. 24). That is, a fusion vector of a human GSK3-derived domain with GFP was constructed using primers of SEQ ID NOS: 23-25 in a manner similar to the method described in Example 10.

GFP-ABL-F (SEQ ID NO: 23): 5'-atg gac gag ctg tac aag ggt acctta eta gga caa cca ata GFP-ABL-R (SEQ ID NO: 24): 5'-tat tgg ttg tec tag taa ggt ace ctt gta cag etc gtc cat ABL-R (SEQ ID NO: 25): 5'-cct gee gcc tct aga ate ctt agt cca agg atg

Whether the constructed expression vectors effectively inhibits the GSK3 nuclear export function of Axin, was examined according to the above-described method.

Among these vectors, the domain proved to have the effects of reducing the expression of Snail and inhibiting the invasive growth of cancer cells is useful for the construction of a polypeptide for inhibiting the export of GSK3 or is useful for the construction of an expression vector for gene therapy. When the expression vector or its fragment is inserted into a conventional gene carrier such as retrovirus, it can be used as a gene therapy agent for treating diseases which occur due to a decrease in the concentration or expression of GSK3 in cells. Also, when the polypeptide bound to nano-sized materials or quantum dots (Q- dots) is administered into cells, it can be used as an agent for treating diseases which occur due to a decrease in the concentration or expression of GSK3 in cells.

Example 13: Construction of nano-sized materials for increasing expression of GSK3 in nucleus

Nanobeads or nanocrystals, which can bind specifically to GSK3 or Axin so as to effectively inhibit the GSK3 nuclear export function of Axin, were constructed. The polypeptide according to the present invention was conjugated to commercially available nanocrystals (Q-dot Corp., USA) according to the method shown in FIG. 25 and were administered into cells. The Q-dots administered into cells bind specifically to GSK3 present in the cells, and real time images can be obtained by tracing the Q-dots using a real time live cell imaging system (Delta Vision RT, USA) (FIG. 26). As a result, it could be seen that the Q-dots fused with TAT entered the cells and were bound to GSK3. When the Q-dots were traced in real time, the intracellular migration of the Q-dots bound to GSK3 can be observed in real time as shown in FIG. 26.

When this real time live cell imaging system is used, kinetics of cells can be traced in real time, and cancer cells or the like can be distinguished from normal cells. Thus, the nanosized materials or quantum dots (Q-dots), having the inventive polypeptide bound thereto, are useful as diagnostic agents for diagnosing diseases, such as cancers, which occur due to a decrease in the concentration of GSK3 in the nucleus. INDUSTRIAL APPLICABILITY

As described in detail above, the present invention provides a cell-permeable polypeptide for inhibiting the development and progression of diseases, which occur due to a decrease in the concentration of GSK3 in the nucleus. When the polypeptide according to the present invention is administered in vivo, it inhibits the GSK3 nuclear export function of Axin and increases the concentration of

GSK3 in the nucleus. Accordingly, the polypeptide according to the present invention is useful for inhibiting the development and progression of various diseases which occur due to a decrease in the concentration of GSK3.

Although a specific embodiment of the present invention has been described in detail, those skilled in the art will appreciate that this description is merely a preferred embodiment and is not construed to limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the accompanying claims and equivalents thereof.

Claims

THE CLAIMSWhat is Claimed is:

1. A polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, which comprises:

(a) a protein transduction domain (PTD), which enables a protein to permeate through a cell membrane without the aid of a cell membrane receptor; and (b) a GSK3 (glycogen synthase kinase 3)-binding domain (GBD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to GSK3.

2. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 1 , which additionally comprises (c) a HA2 domain which can avoid a decrease in biological activity, which is caused by macropinosome or endosome.

3. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 1, which additionally comprises a nuclear localization signal

(NLS) for inhibiting export of GSK3 from nucleus to cytoplasm.

4. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 1 , wherein the PTD is TAT.

5. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 1 , wherein the GBD is an Axin-derived, GSK3-binding domain, which inhibits the binding of GSK3 to Axin.

6. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 1 , wherein the GBD is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7.

7. A recombinant vector, comprising: a base sequence encoding a protein transduction domain (PTD), which enables a protein to permeate through the cell membrane without the aid of a cell membrane receptor; and a base sequence encoding a GSK3 (glycogen synthase kinase 3)-binding domain (GBD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to GSK3.

8. The recombinant vector according to claim 17, which additionally comprises a base sequence encoding a HA2 domain which can avoid a decrease in biological activity, caused by macropinosome or endosome; or a base sequence encoding a nuclear localization signal (NLS) for inhibiting the export of GSK3 from nucleus to cytoplasm.

9. A recombinant microorganism transformed with the recombinant vector of claims 7 or 8.

10. A method for preparing a polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, the method comprising: culturing the recombinant microorganism of claim 9; and recovering said polypeptide.

11. A polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, which comprises

(a) a protein transduction domain (PTD), which enables a protein to permeate through a cell membrane without the aid of a cell membrane receptor; and

(b) an Axin-binding domain (ABD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to Axin.

12. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 11 , which additionally comprises (c) a H A2 domain which can avoid a decrease in biological activity, which is caused by macropinosome or endosome.

13. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 11 , which additionally comprises a nuclear localization signal (NLS) for inhibiting export of GSK3 from nucleus to cytoplasm.

14. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 11 , wherein the PTD is TAT.

15. The polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm according to claim 11, wherein the ABD is a GSK3-derived, Axin-binding domain, which inhibits the binding of GSK3 to Axin.

16. A recombinant vector, comprising: a base sequence encoding a protein transduction domain (PTD), which enables a protein to permeate through a cell membrane without the aid of a cell membrane receptor; and a base sequence encoding an Axin -binding domain (ABD), which serves to inhibit the export of GSK3 from nucleus to cytoplasm through binding to Axin.

17. The recombinant vector according to claim 16, which additionally comprises a base sequence encoding a HA2 domain which can avoid a decrease in biological activity, caused by macropinosome or endosome; or a base sequence encoding a nuclear localization signal (NLS) for inhibiting export of GSK3 from nucleus to cytoplasm.

18. A recombinant microorganism transformed with the recombinant vector of claims 16 or 17.

19. A method for preparing a polypeptide for inhibiting export of GSK3 from nucleus to cytoplasm, the method comprising: culturing the recombinant microorganism of claim 18; and recovering said polypeptide.

20. A pharmaceutical composition for inhibiting the growth and metastasis of cancer cells, the composition comprising the polypeptide of any one claim among claims 1~3 and claims 11-13, as an active ingredient.

21. A pharmaceutical composition for treating immune diseases, the composition comprising the polypeptide of any one claim among claims 1-3 and claims 11-13, as an active ingredient.

22. The pharmaceutical composition according to claim 21, wherein said immune disease is arthritis.

23. A formulation for treating diseases which occurs due to a decrease in the concentration of GSK3 in the nucleus, the formulation comprising the polypeptide of any one claim among claims 1—3 and claims 1 1-13 bound to nano-sized materials or quantum dots (Q-dots).

24. The formulation for treating diseases according to claim 23, wherein the diseases occurring due to a decrease in the concentration of GSK3 in the nucleus is cancers or immune diseases.

25. A gene therapy agent for treating diseases occurring due to a decrease in the concentration of GSK3 in the nucleus, which contains: a therapeutic gene encoding the polypeptide of any one claim among claims 1-3 and claims 11-13; and a carrier thereof.

26. The gene therapy agent according to claim 25, wherein the carrier is a viral vector.

27. The gene therapy agent according to claim 25, wherein the diseases occurring due to a decrease in the concentration of GSK3 in the nucleus is cancers or immune diseases.

28. A diagnostic agent for diagnosing diseases which occur due to a decrease in the concentration of GSK3 in the nucleus, the diagnostic agent comprising the polypeptide of any one claim among claims 1-3 and claims 1 1-13 bound to nano-sized particles or quantum dots (Q-dots).

29. The diagnostic agent for diagnosing diseases according to claim 28, wherein the diseases occurring due to a decrease in the concentration of GSK3 in the nucleus is cancers or immune diseases.

30. A method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, the method comprising:

(a) blocking the Axin-binding domain of GSK;

(b) blocking the GSK3-binding domain of Axin;

(c) inhibiting the activation of Axin; or

(d) increasing the expression of GSK3 in the nucleus.

31. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, wherein said (a) blocking the Axin-binding domain of GSK3 is performed using a compound, a peptide or a polypeptide, which bind to the Axin-binding domain of GSK3.

32. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, according to claim 31 , wherein said polypeptide is the polypeptide of any one claim among claims 1-3.

33. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, according to claim 30, wherein said (b) blocking the GSK3-binding domain of Axin is performed using a compound, a peptide or a polypeptide, which bind to the GSK3-binding domain of Axin.

34. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, according to claim 33, wherein said polypeptide is the polypeptide of any one claim among claims 11-13.

35. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, according to claim 30, wherein said (c) inhibiting the activation of Axin is performed by inhibiting Wnt signaling in a Wnt//3-catenin pathway or inhibiting the activity of (3-catenin.

36. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, according to claim 30, wherein the method is carried out using the formulation of claim 32.

37. The method for inhibiting a decrease in the concentration of GSK3 in the nucleus, caused by the nuclear export of GSK3 through binding to Axin, according to claim 30, wherein the method is carried out using said gene therapy agent of claim 34.