KR100986345B1 - Screening Methods of Anticancer Agent Using SUMOylation of Pontin - Google Patents

Abstract

The present invention relates to a method for screening an anticancer agent using pontin hydration, and provides a methionized pontin linked to SUMO protein to 225th lysine of pontin and screens an anticancer agent using the fontin. Provide a way to. More specifically, culturing the cells; Contacting the cell with a potential substance; Confirming that the intracellular pontin hydration decreases as compared to the control (test without contacting the cell with the potential material); And it provides a method for screening anticancer drugs comprising the step of selecting the latent substance to reduce the hydration of the pontin as an anticancer agent. In another aspect, the present invention comprises the steps of preparing a solution containing pontin and proteins necessary for the hydration (a hair activating enzyme, a hair Binding enzyme, a hair ligase) and ATP; Treating the potential material with the solution; Confirming that the hydroponification of pontin in the solution decreases relative to the control (test without contacting the potential material with the solution); And it provides a method for screening anticancer drugs comprising the step of selecting the latent substance to reduce the hydration of the pontin as an anticancer agent.

Pontin, hydration, anticancer agent

Description

Screening Methods of Anticancer Agent Using SUMOylation of Pontin}

The present invention relates to a method for screening an anticancer agent using SUMOylation of pontin.

Identifying signal integration mechanisms involving various molecular mechanisms at the level of gene transcription is an important goal in biology. The main downstream effects of the signaling pathway are the regulation of the function of transcriptional and coregulators in the nucleus (1, 2). Therefore, it is important to study the role of transcription factors and co-regulators in responding to upstream signaling pathways. Androgen receptor ( AR ) not only regulates prostate development but also acts as a major regulator of primary prostate cancer growth (3, 4). The effect of androgens is modulated through the androgen receptor, which is one of a family of nuclear receptors that function as ligand-induced transcription factors that regulate the expression of target genes with androgen response elements. Similar to other nuclear receptor subfamilies, the androgen receptor contains a DNA binding domain and a C-terminal ligand binding domain that function as ligand dependent nuclear receptors with transcriptional activation domains AF-1 and AF-2 (5, 6). ). The ability of many nuclear receptors to bind to various ligands, including agonists or antagonists, to enhance or decrease gene activity is modulated by coactivators and corepressors. We present interesting issues about the combined transcription mechanism.

Gene expression is affected by chromatin structure, and covalent modification of histones plays an important role in regulating transcription and chromatin kinetics (7, 8). Transcription of most genes is regulated by the coordinate action of the chromatin-reconstituted complex. Pontin and leptin are components of the ATP dependent chromatin-reconstituting complex and have been reported to be closely associated with the bacterial DNA helicase RuvB (9, 10). Fontin is known to bind and activate β-catenin / T cell factor ( TCF ) transcriptional complexes, while leptin is known to inhibit the Wnt / β-catenin signaling pathway (11). In mammals, pontin and leptin become components of the Tip60 coactivator complex with intrinsic histone acetyltransferase activity (12). Leptin / pontin ratios in zebrafish embryos regulate cardiac growth during development by the β-catenin pathway (13).

Post-transcriptional modification of proteins plays an important role in the functional regulation of transcriptional co-regulators. Many enzymatic activities have been known to be associated with co-regulatory complexes including histone acetylation / deacetylation, phosphorylation / dephosphorylation, ubiquitination and SUMOylation (14, 15). Ubiquitin similar small changes proteins (small ubiquitin-like modifier; la SUMO, the "fine animal ") is an important regulatory role in many cellular processes, including signal transduction, transcriptional regulation, chromatin structure and nuclear / cytoplasmic transport ( 15-18). Medulla is a protein of approximately 11 kDa, structurally similar to ubiquitin but not functionally. The enzymatic process of adding and removing hair protein to the target protein differs from the ubiquitination process. Hydromyelination of the target protein was performed by the E-1 activation molecules SAE1 / SAE2 in animal cells; E-2-conjugation enzyme Ubc9; And E3 ligase (19). Several hair-processing enzymes have been found to hydrolyze the hairy part from target substrates in stray and yeast (20-22). Unlike ubiquitination, it is not known that hair modifications are associated with proteolysis. Rather, it is similar to the non-proteolytic role of ubiquitination, including the regulation of subcellular localization and transcriptional activity. A large number of hair modification proteins are known, including RanGAP1, p53, c-Jun and leptin (23-26). Lysine appears as a point for various modifications, which is expected to have complex modification codes in both histone and nonhistone proteins.

Recently we reported the kinetics of the chromatin reconstitution complex in the regulation of KAI1 , a mutation suppressor gene (25, 27). The leptin chromatin reconstitution complex contains SENP1 dehydrogenase as a constituent, and the medulla modification of leptin plays an important role in the regulation of transcription of KAI1 (25). In the present invention, the pontin chromatin reconstitution complex is modified by hair, but this modification has been found to promote differential function regulation of other down target genes compared to leptin. Biochemical separation of the pontin containing the complex indicated that the Ubc9 hydrozygotease was the binding partner. In the regulation of androgen receptor target genes, such as the prostate-specific antigen ( PSA ) gene, in prostate cancer cells, mutations affecting the binding of pontin and hairs reduced the transcriptional activation function of pontin. Hydroponification of pontin is associated with active regulatory mechanisms, which relate to the transcriptional activation function of pontin through synergy of retention and coactivator binding in the nucleus. On the other hand, lemonogenesis of leptin is associated with enhanced transcription inhibition. Moreover, pontin medification increased the proliferation and growth of prostate cancer cells. Taken together, the inventors for the first time have discovered a molecular mechanism for the hair modification of pontin chromatin reconstitution complexes involved in transcriptional regulation, based on the fact that the androgen receptor target genes involved in cancer progression are strongly activated.

International Patent Application No. PCT / US2006 / 041084 (First date 2005. 10. 21) is a therapeutic composition for the prevention and treatment of neurodegenerative diseases (Parkinson's disease, Huntington's disease, Alzheimer's disease, Kennedy's disease and spinal cord cerebellar ataxia) And a screening method thereof. However, there is no mention of a method for screening an anticancer drug using pontin hydration.

An object of the present invention is to provide a method for effectively screening an anticancer agent using the hydroponification of pontin.

In order to achieve the above object, the present invention provides a summed pontin linked to SUMO protein in the 225th lysine of pontin.

The present invention provides a method for screening an anticancer agent using the above hydrous pontin. More specifically, culturing the cells; Contacting the cell with a potential substance; Confirming that the intracellular pontin hydration decreases as compared to the control (test without contacting the cell with the potential material); And it provides a method for screening anticancer drugs comprising the step of selecting the latent substance to reduce the hydration of the pontin as an anticancer agent. Preferably the cancer is prostate cancer. The cell is characterized in that the cancer cell. Preferably the cancer cells are characterized in that the prostate cancer cells, more preferably the prostate cancer cells are characterized in that the LNCaP cells.

The method for confirming the reduction of pontin hydration is characterized by confirming the expression of an androgen receptor or a retinoid related orphan receptor ( ROR ) promoter, and the androgen receptor or retinoid The method of confirming the expression of a Reninoid related orphan receptor ( ROR ) promoter provides an anticancer screening method characterized by confirming the expression of a reporter functionally linked with an androgen receptor or a retinoid related orphan receptor promoter.

The present invention comprises the steps of preparing a solution containing pontin and proteins necessary for hydration (hair activating enzyme, hair Binding Enzyme, hair ligase) and ATP; Treating the potential material with the solution; Confirming that the hydroponification of pontin in the solution decreases relative to the control (test without contacting the potential material with the solution); And it provides a method for screening anticancer drugs comprising the step of selecting the latent substance to reduce the hydration of the pontin as an anticancer agent. The method for confirming pontin hydrolysis is an immunochemical method, a method using a radioisotope material, a method based on molecular weight difference according to electrophoresis, and a method using a fluorescent dye. More specifically, the immunochemical method is characterized by using an ELISA.

A first aspect of the present invention provides a heminized pontin linked to SUMO protein at the 225th lysine of pontin. The pontin has a nucleotide sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2. Respective NCBI access numbers are AF099084 and AAD04427.

The term "hair" as used herein is a ubiquitin-like small ubiquitin-like modifier ( SUMO ), a protein of 97 amino acids. The hairs have a nucleotide sequence of SEQ ID NO: 3 and an amino acid sequence of SEQ ID NO: 4. Each NCBI access number is NM_001005781 and NP_001005781.

As used herein, the term "SUMOylation" refers to the formation of isopeptide bonds of ε-amino groups at the C-terminal carboxyl group and pontin 225th lysine residues of the meric protein.

A second aspect of the present invention provides a method for screening an anticancer agent using the above-mentioned hydrocyclized pontin. More specifically, culturing the cells; Contacting the cell with a potential substance; Confirming that the intracellular pontin hydration decreases as compared to the control (test without contacting the cell with the potential material); And it provides a method for screening anticancer drugs comprising the step of selecting the latent substance to reduce the hydration of the pontin as an anticancer agent. Preferably the cancer is prostate cancer.

The cell is characterized in that the cancer cell. Preferably the cancer cells are characterized in that the prostate cancer cells, more preferably the prostate cancer cells are characterized in that the LNCaP cells.

The method for confirming the reduction of pontin hydration is characterized by confirming the expression of an androgen receptor or a retinoid related orphan receptor ( ROR ) promoter, and the androgen receptor or retinoid The method of confirming the expression of a Reninoid related orphan receptor ( ROR ) promoter provides an anticancer screening method characterized by confirming the expression of a reporter functionally linked with an androgen receptor or a retinoid related orphan receptor promoter.

The reporter is not limited to a particular gene. Any gene that expresses a product that can be easily measured can be used. Suitable reporter genes are well known to those skilled in the art. Examples of reporter genes include other enzyme detection systems such as CAT (chloramphenicol acetyl transferase), luciferase, beta-galactosidase; Bacterial luciferase, alkaline phosphatase and green fluorescent material (GFP) are included, but are not limited to these. Detection of reporter genes is well known to those skilled in the art. When a reporter gene is detected by its enzymatic activity, it generally comprises providing the enzyme with its appropriate substrate and detecting the reaction product (eg light produced by luciferase). Detection can include simply detecting the presence or absence of the reporter gene product. Another method may include quantification of the expression level of the reporter gene product. The method of quantification can be absolute quantification or can be quantified compared to the expression level of the housekeeping gene. Such detection may be performed manually or may be performed automatically as in a high-throughput screening system. Preferably the reporter is luciferase.

A third aspect of the present invention comprises the steps of preparing a solution containing pontin and proteins necessary for hydration (hair activating enzyme, hair Binding Enzyme, hair ligase) and ATP; Treating the potential material with the solution; Confirming that the hydroponification of pontin in the solution decreases relative to the control (test without contacting the potential material with the solution); And it provides a method for screening anticancer drugs comprising the step of selecting the latent substance to reduce the hydration of the pontin as an anticancer agent.

The SUMO activating enzyme (SUMO activating enzyme) is the end of the carboxyl group of the hair to attack the ATP to produce pyrophosphate and hair-adenylate, the cysteine of the enzyme active site to attack the hair-adenylate to enzyme and hair Allow high energy thioester bonds to be created between.

The SUMO conjugation enzyme (SUMO conjugation enzyme) is responsible for the process of delivery of the hair to the lysine residues of pontin. There is only one Ubc9 for hydrophilization.

The SUMO ligases form a complex with Ubc9 and serve to move to the nucleus.

The method for confirming pontin hydrolysis is an immunochemical method, a method using a radioisotope material, a method based on molecular weight difference according to electrophoresis, and a method using a fluorescent dye. More specifically, the immunochemical method is characterized by using an ELISA.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Pontin Chromatin  Isolation of Reconstituted Complexes

We previously reported the role of leptin chromatin reconstitution complexes in regulating KAI1 expression (25). Although leptin and pontin are structurally similar, they are expected to play distinctly different roles in terms of target gene regulation. To find other functions of pontins, we used a flag epitope-tag strategy to isolate complexes containing pontins. To identify pontin complex proteins isolated from the Flag M2 affinity column, liquid chromatography (LC-MS / MS) coupled with tandem mass spectrometry was used ( FIG. 1A and FIG . 1 ) . 1B ). LC-MS / MS analysis confirmed the presence of leptin, β-catenin, and p400, well known binding partners for pontin (11, 28, 29), and these results illustrate the functional association between these molecules.

Another protein identified by LC-MS / MS analysis is the hair-conjugation enzyme Ubc9 ( FIGS. 1A and 1B ). Association of pontin with these polypeptides was confirmed by immunoblotting analysis of the eluate ( FIG. 1C ). To further confirm the association between pontin and Ubc9, an endogenous co-immunoprecipitation assay was performed. This showed that pontin specifically binds to Ubc9 ( FIG. 1D ). By identifying the hairpin-conjugating enzymes in the pontin-containing complex, we tested whether hairpinning modifications affected the functional regulation of pontin.

< Example  2> Pontin Mutation  And Mutated Pontin Confiscation

1. Reagent

Antibodies of anti β-catenin, p400, Ubc9 and SUMO were purchased from Santa Cruz Biotechnology. 5α-dihydroxytestosterone ( DHT ) was purchased from Sigma. Lipofectamine 2000 reagent was used for transfection experiments and was purchased from Invitrogen.

2. Intracellular Confiscation  analysis( In vivo SUMOylation assay )

293T cells were transfected with pcDNA-fontin-TAP and His ⁶ -SUMO1. After 36 hours of transfection, lysis buffer (150 mM NaCl, 25 mM Tris) with the addition of a complex protease inhibitor cocktail (Beringer Mannheim) and 0.2% sodium dodecyl sulfate ( SDS ) Cells were lysed with -HCl, pH 7.8, 0.1% Nonidet P40, and 1 mM EDTA), sonicated for a while and centrifuged. The clear extract was added 15 ul Ni-NTA agarose beads (Ni-NTA agarose beads, Qiagen) and incubated for 2 hours. The slurry was washed with lysis buffer. After denaturation, the proteins were separated by SDS-polyacrylamide gel electrophoresis ( PAGE ) and the presence of pontin was confirmed by immunoblotting.

3. In vitro Confiscation  analysis( In in vitro SUMOylation assay )

In vitro hematuration assays were performed using 1 ug of recombinant pomace protein, 150 ng of E1 (SAE1 / SAE2), 10 ng of E2 (Ubc9), and 1 ul of prepared 35S-methionine-labeled in vitro-translated pontin ( ATP regeneration system with 35S-methionine-labeled in vitro-translated pontin (50 mM Tris-HCl, pH 7.6, 5 mM MgCl ² , 2 mM ATP, 10 mM creatine phosphate, 3.5 U / ml creatine kinase) TNT T7 Quick-coupled reticulocyte lysate kit (Promega) from 10 ul reactant containing (eatine kinase) and 0.6 U / ml inorganic pyrophosphatase] It was performed using.

4. Indirect Immunofluorescence Assay

HeLa cells were cultured on coverslips and washed three times with phosphate-buffered saline ( PBS ). Cells were then fixed for 30 minutes at room temperature in 2% paraformaldehyde dissolved in PBS, washed with PBS and permeabilize for 30 minutes at room temperature with 0.5% Triton X-100 ( PBST ) dissolved in PBS. ). Blocking was performed for 30 minutes with 10% gelatin dissolved in 3% horse serum and PBST. For staining, affinity-purified anti-Flag M2 IgG was added and the cells were incubated for 1 hour. Fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Immuno Research Lab) were added and the stained cells were incubated for 2 hours and washed three times with PBST.

5. Results

We established an in vitro hair modification system to see if pontin could be a substrate for the hair-conjugation enzyme Ubc9 (30, 31). Case when the detached fine animal addition or not added, the separated E1 (SAE2 / AOS2) and E2 and ³⁵ S- labeled in an animal hair Chemistry mixed solution containing the (Ubc9) in vitro translated (in vitro- translated) pontin protein was reacted. The pontin conjugate was formed after the addition of hydroceps to the hydrophilized mixed solution ( FIG. 2A ). These results indicated that pontin had a hair modification. In order to identify the functional lysine residues of pontin that act as the hair receptor site, we found the consonant consensus sequence, ψ KxE (, ψ represents many hydrophobic amino acids) ( FIG. 2B ) (32). Each pontin 6 lysine was independently converted to arginine, and the resulting mutants were tested for in vitro and intracellular hydrocephalus modification ability ( FIG. 2C , FIG. 2D ). K2R, K108R, K171R, K231R and K268R mutants showed little or little change in the pontine's hair modification. On the other hand, the K225R mutant did not undergo modification by pontin hair growth both in vitro and in cells ( FIG. 2C , FIG. 2D ). Thus, only the K225 single lysine residue of pontin is shown to function as a modulatory receptor site.

Then, in order to eliminate the problems caused by overexpressed hair growth or indirect effects associated with Ubc9, we made constant pontin in the hematurated form and confirmed the expression through immunoblot analysis ( FIG. 2E ). Since medulization has been known to regulate nuclear / cytoplasmic transport of many transcription and co-regulatory factors (33, 34), we tested whether the pontin conjugation plays a role in translocation between the nucleus and the cytoplasm. While pontin localizes both in the nucleus and cytoplasm, pontin K225R mutants appear to have more cytoplasm localization than wild type pontin ( FIG. 2F ). On the other hand, the concomitant binding of wild type pontin and pontin K225R mutants showed exclusive nuclear localization results. These results demonstrate that covalent modifications by the hair shaft through the increased nuclear retention of pontins demonstrate pontin's strong transcriptional control of the target gene set in the nucleus.

< Example  3> Pontin  Hair deformation Androgens  Effect on transcriptional activation of receptor target genes

Through TOPFLASH reporter assay, it is already known that pontin activates β-catenin-mediated transcriptional activity while leptin inhibits β-catenin-mediated transcriptional activity (11). Pontine is collected in the KAI1 promoter with Tip60 coactivator, but KAI1 It is not necessary for transcriptional activation. On the other hand, leptin, along with β-catenin, plays a critical role in inhibiting KAI1 (27). Transcriptional regulation by androgen receptors involves interaction with various transcriptional coactivators in the presence of agonists, which act in both continuous and combinational methods (35). β-catenin and Ubc9 were reported to function as androgen receptor coactivators (36), and their association with pontin was confirmed from the pontin-containing complex shown in FIG. 1C. We tested whether pontin affects 5α-dihydroxytestosterone ( DHT ) -dependent androgen receptor target gene activity. Prostate-specific antigen ( PSA ), KLK2 using reverse transcriptase ( RT ) -PCR analysis And NKX3 .1 To evaluate the effect of shRNA- mediated knockdown of osteopontin (knockdown) for DHT- induced transcriptional activation of the androgen receptor, such as the target gene (Figure 3A). Quantitative RT-PCT analysis reaffirmed the effect of pontin knockdown on DHT-dependent transcriptional activation of androgen receptor target genes ( FIG. 3B ). Knockdown of leptin did not affect the expression of androgen receptor target genes (data not shown). This led to the finding that pontin is required for the activation of DHT-dependent androgen receptor target genes.

Pontin and leptin are closely related to ATPase related elements with various cellular activities and are components of many different complexes, including Tip60, IN080 and p400 complexes involved in transcriptional regulation (9, 12, 28). To obtain information about the role of pontin's hair modification, we evaluated the effects of the hair modification on the transcriptional properties of pontin. The introduction of pontin K225R reduced androgen receptor gene transcripts, and the constant, hydrophilized, hair-bound pontin K225R activated the androgen receptor target gene transcript when DHT was added ( FIG. 3C ). As shown in the consistent results of FIG. 3C, expression of pontin K225R mutants inhibited the activation function of pontin in the androgen responsive element ( ARE ) -luciferase reporter when DHT was added, whereas the hair-bound The pontin K225R mutant further activated the ARE-luciferase reporter ( FIG. 3D ). In addition, the expression of pontin activates the Retinoid-related orphan receptor α2 element ( ROR α2E ) -luciferase reporter ( FIG. 3E ), but the retinoic acid response element ( RARE ) or estrogen The response response element ( ERE ) -luciferase reporter is not activated ( FIG. 3F and data not shown). Immunoprecipitation results confirmed that pontin binds to androgen receptor (AR) and retinoid related orphan receptor α2 (RORα2) but not to retinoic acid receptor (RAR) and estrogen receptor (ER) ( FIG. 3G and data not shown). Not). These results indicate that pontin is specifically involved in nuclear receptor regulatory transcriptional function and that the pontine conjugation is involved in the mechanism by which pontin regulates transcriptional activation.

In most cases, hair modifications of certain transcription factors and co-regulators have the potential for transcriptional inhibition (25, 32). The hemophilized leptin binds to histone deacetylase 1 ( HDAC1 ) more strongly than unmyelinated leptin, which explains why the hydrophilic modification of leptin increases leptin's transcriptional suppression function ( 25). In order to test whether the hair modification of pontin alters the binding affinity with other transcription cofactors that modulate and enhance transcriptional activity, hair bound pontin was specifically bound to androgen receptor co-transcriptors ( FIG. 3H). ). While wild-type pontin and pontin K225R mutants bound to Tip60 coactivators, pontin K225R mutants were highly compatible with β-catenin and cAMP regulatory element-binding protein ( CBP ) coactivators compared to wild-type pontin. Weakly bound ( FIG. 3H ). These results show that pontin hair modifications are important for maintaining and exerting coactivator mediated androgen receptor transcriptional activating functions.

< Example  4> Pontin Conqueror  Effect on proliferation and growth of prostate cancer cells

1. Chromatin immunoprecipitation ( ChIP )

Chromatin immunoprecipitation assays were performed as previously reported using truncated fragments between about 300 bps and 1 kb in size (27). For PCR experiments, 1 ul to 30 ul of DNA extract was used and amplified about 25-30 times. For a two-step chromatin immunoprecipitation assay, the compositions were eluted from the first immunoprecipitation reaction by reaction with 10 mM dithiothreitol ( DTT ) at 37 ° C. for 30 minutes and using chromatin immunoprecipitation dilution buffer 1. Dilution was performed at a: 50 ratio and a reimmune sedimentation assay was performed with the secondary antibody. The composition of the chromatin immunoprecipitation dilution buffer is 20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid ( EDTA ) and 1% Triton X-100.

2. Cell Transformation Assay

Adhesion-independent growth of mock-, pontin-, pontin-K225R-, sumo-pontin- and sumo-pontin K225R-expressing LNCaP cells may inhibit cell growth in semisolid medium. Was determined by analysis. Cells 10 ⁵ were placed in Iscove's media containing 0.4% noble agar with 10% FCS. Cells were allowed to grow for 3 weeks at 5% CO and analyzed for the formation of colonies containing> 50 cells.

3. Results

Androgens promote prostate cell growth, and LNCaP cell growth is inhibited by androgen deficiency (37). To test if androgens affect pontin hydration, and to determine the potential effects of pontin hydration on cancer cell growth and proliferation, add 5α-dihydroxytestosterone ( DHT ) and Intracellular pluripotency analysis of pontin was performed when not added ( FIG. 4A ). Interestingly, DHT treatment significantly increased pontin's hydrophilization. Next, it was tested for the presence of vesinized pontin in the prostate-specific antigen ( PSA ) promoter under active conditions. In order to monitor whether hematurated pontin was present in the PSA promoter when DHT was added, a two step chromatin immunoprecipitation assay ( ChIP ) was performed ( FIG. 4B ). Water soluble chromatin from DHT treated LNCaP cells was immunoprecipitated with anti-pontin antibodies isolated from immune complexes and reimmunized with anti-heading antibodies. Through this experiment, there was a fine animal a modified osteopontin under active conditions can actually perform quite immunoprecipitation method PSA promoter, indicating that the presence of PSA promoter (Fig. 4B). Since Ubc9 is a single hydrophilic conjugation enzyme and the interaction of Ubc9 with the androgen receptor DNA binding domain and hinge region in yeast has been reported (36), it was tested whether Ubc9 gathered at the PSA promoter for transcriptional activation. Chromatin immunoprecipitation assay showed that Ubc9 actually gathered in the PSA promoter when DHT was added ( FIG. 4C ), and knockdown of Ubc9 resulted in PSA And androgen receptor target transcripts such as KLK2 were reduced ( FIG. 4D ). This indicates that Ubc9 is required for transcriptional activation of androgen receptor target genes.

Hydroponification of pontin is prostate-specific antigen ( PSA ) To determine whether gene activation is necessary and whether it can be expanded physiologically appropriately to prostate cancer cells, the effect of pontin on the proliferation and growth of LNCaP cells was examined. Proliferation assays were performed by measuring the increase in cell numbers over 5 days of pontin K225R-, sumo-pontin- and sumo-pontin K225R-expressing LNCaP cells against mock cells. The introduction of pontin K225R mutants inhibited the growth and proliferation of LNCaP cells, but the sumo-pontin and sumo-pontin K225R promoted cell proliferation ( FIG. 4E ). Taken together, these results indicate that recruitment of the congested pontin to the androgen receptor affects the androgen's cell proliferative capacity in prostate cancer.

To determine if pontin hydration promotes adhesion-independent growth, we tested soft agar medium colony formation of LNCaP cells stably expressing pontin, pontin K225R, sumo-pontin and sumo-pontin K225R, This is an important characteristic of cancer cell growth ( FIG. 4F ). The introduction of sumo-pontin greatly improved the size of the colonies. Colonies with diameters greater than 250 μm were not observed in mock LNCaP cells and cells overexpressing pontin K225R, but were observed in sumo-pontin and sumo-pontin K225R cells. These results indicate that, as with the in vitro androgen receptor dependent transcriptional activation results, the hair modification of pontin can increase the transgenic ability of pontin.

The present invention is a screening method that can efficiently search for an anticancer agent using the hydroponification of pontin. The hydration modification and hydration state of pontin protein are physiologically important because they can affect various phenomena such as cancer-related signaling, transcription regulation, and cell growth regulation. Controlling the attachment and dropping of pontin proteins can affect cancer cell proliferation and growth, which can be used to screen anticancer drugs and develop new anticancer drugs. In addition, it can be applied to the development of therapeutics for a variety of diseases based on the mechanism of the hydration of proteins associated with various diseases.

[REFERENCES]

1.Glass, CK & Rosenfeld, MG (2000) Genes Dev 14, 121-141.

McKenna, NJ &O'Malley, BW (2002) Cell 108, 465-474.

Feldman, BJ & Feldman, D. (2001) Nat Rev Cancer 1, 34-45.

4. Heinlein, CA & Chang, C. (2004) Endocr Rev 25, 276-308.

5.Schaufele, F., Carbonell, X., Guerbadot, M., Borngraeber, S., Chapman, MS, Ma, AA, Miner, JN, & Diamond, MI (2005) Proc Natl Acad Sci USA 102, 9802-9807.

6.Shaffer, PL, Jivan, A., Dollins, DE, Claessens, F., & Gewirth, DT (2004) Proc Natl Acad Sci USA 101, 4758-4763.

Berger, SL (2007) Nature 447, 407-412.

Jenuwein, T. & Allis, CD (2001) Science 293, 1074-1080.

Jonsson, ZO, Jha, S., Wohlschlegel, JA, & Dutta, A. (2004) Mol Cell 16, 465-477.

10.Shen, X., Mizuguchi, G., Hamiche, A., & Wu, C. (2000) Nature 406, 541-544.

Bauer, A., Chauvet, S., Huber, O., Usseglio, F., Rothbacher, U., Aragno, D., Kemler, R., & Pradel, J. (2000) EMBO J 19, 6121. -6130.

12.Ikura, T., Ogryzko, VV, Grigoriev, M., Groisman, R., Wang, J., Horikoshi, M., Scully, R., Qin, J., & Nakatani, Y. (2000) Cell 102, 463-473.

13. Rottbauer, W., Saurin, AJ, Lickert, H., Shen, X., Burns, CG, Wo, ZG, Kemler, R., Kingston, R., Wu, C., & Fishman, M. ( 2002) Cell 111, 661-672.

14.Baek, SH & Rosenfeld, MG (2004) Biochem Biophys Res Commun 319, 707-714.

15. Kerscher, O., Felberbaum, R., & Hochstrasser, M. (2006) Annu Rev Cell Dev Biol 22, 159-180.

16.Baek, SH (2006) Cell Cycle 5, 1492-1495.

17.Kim, KI, Baek, SH, & Chung, CH (2002) J Cell Physiol 191, 257-268.

18.Kirkin, V. & Dikic, I. (2007) Curr Opin Cell Biol 19, 199-205.

19.Kim, KI & Baek, SH (2006) Mol Cells 22, 247-253.

20. Bylebyl, GR, Belichenko, I., & Johnson, ES (2003) J Biol Chem 278, 44113-44120.

Lee, MH, Lee, SW, Lee, EJ, Choi, SJ, Chung, SS, Lee, JI, Cho, JM, Seol, JH, Baek, SH, Kim, KI , et al . (2006) Nat Cell Biol 8, 1424-1431.

Mikolajczyk, J., Drag, M., Bekes, M., Cao, JT, Ronai, Z., & Salvesen, GS (2007) J Biol Chem .

Bossis, G., Malnou, CE, Farras, R., Andermarcher, E., Hipskind, R., Rodriguez, M., Schmidt, D., Muller, S., Jariel-Encontre, I., & Piechaczyk , M. (2005) Mol Cell Biol 25, 6964-6979.

24.Feng, L., Lin, T., Uranishi, H., Gu, W., & Xu, Y. (2005) Mol Cell Biol 25, 5389-5395.

25.Kim, JH, Choi, HJ, Kim, B., Kim, MH, Lee, JM, Kim, IS, Lee, MH, Choi, SJ, Kim, KI, Kim, SI , et al . (2006) Nat Cell Biol 8, 631-639.

26.Sternsdorf, T., Jensen, K., & Will, H. (1997) J Cell Biol 139, 1621-1634.

27.Kim, JH, Kim, B., Cai, L., Choi, HJ, Ohgi, KA, Tran, C., Chen, C., Chung, CH, Huber, O., Rose, DW , et al . (2005) Nature 434, 921-926.

28.Fuchs, M., Gerber, J., Drapkin, R., Sif, S., Ikura, T., Ogryzko, V., Lane, WS, Nakatani, Y., & Livingston, DM (2001) Cell 106, 297-307.

Feng, Y., Lee, N., & Fearon, ER (2003) Cancer Res 63, 8726-8734.

30.Tatham, MH, Jaffray, E., Vaughan, OA, Desterro, JM, Botting, CH, Naismith, JH, & Hay, RT (2001) J Biol Chem 276, 35368-35374.

Seeler, JS, Marchio, A., Losson, R., Desterro, JM, Hay, RT, Chambon, P., & Dejean, A. (2001) Mol Cell Biol 21, 3314-3324.

32. Johnson, ES (2004) Annu Rev Biochem 73, 355-382.

33.Chalkiadaki, A. & Talianidis, I. (2005) Mol Cell Biol 25, 5095-5105.

34.Salinas, S., Briancon-Marjollet, A., Bossis, G., Lopez, MA, Piechaczyk, M., Jariel-Encontre, I., Debant, A., & Hipskind, RA (2004) J Cell Biol 165, 767-773.

35.Shang, Y., Myers, M., & Brown, M. (2002) Mol Cell 9, 601-610.

36.Poukka, H., Aarnisalo, P., Karvonen, U., Palvimo, JJ, & Janne, OA (1999) J Biol Chem 274, 19441-19446.

Kokontis, JM, Hay, N., & Liao, S. (1998) Mol Endocrinol 12, 941-953.

38. Metzger, E., Wissmann, M., Yin, N., Muller, JM, Schneider, R., Peters, AH, Gunther, T., Buettner, R., & Schule, R. (2005) Nature 437, 436-439.

39.Yamane, K., Toumazou, C., Tsukada, Y., Erdjument-Bromage, H., Tempst, P., Wong, J., & Zhang, Y. (2006) Cell 125, 483-495.

40. Wang, J., Scully, K., Zhu, X., Cai, L., Zhang, J., Prefontaine, GG, Krones, A., Ohgi, KA, Zhu, P., Garcia-Bassets, I , et al . (2007) Nature 446, 882-887.

41.Mo, YY & Moschos, SJ (2005) Expert Opin Ther Targets 9, 1203-1216.

McDoniels-Silvers, AL, Nimri, CF, Stoner, GD, Lubet, RA, & You, M. (2002) Clin Cancer Res 8, 1127-1138.

43. Mo, YY, Yu, Y., Theodosiou, E., Ee, PLR, & Beck, WT (2005) Oncogene 24, 2677-2683.

1 shows the results of isolation and identification of pontin-containing complexes. (A) Pontin-containing complexes were immunoprecipitated with anti-Flag antibody-conjugated agarose beads from 293T cell extract, binding proteins were eluted with flag peptides and analyzed by SDS-PAGE . (B) The peptide sequence of the pontin related polypeptide from LC-MS / MS analysis was shown to be leptin, β-catenin, p400 and Ubc9, where Ubc9 is a component of the pontin containing complex. (C) Western blot analysis was performed using the indicated antibodies, and leptin, β-catenin, p400 and Ubc9 were detected in the eluate. (D) Pontine interacts with Ubc9. Cell lysates were subjected to immunoprecipitation experiments with anti-Ubc9 IgG or control IgG, and the resulting precipitates were subjected to immunoblotting experiments with anti-pontin IgG. IP : immunoprecipitation, IB : immunoblotting

2 shows the result that lysine 225 residues of pontin are required for the hair modification. (A) In vitro modification of pontin by hair growth is shown. With or without the addition of the hairs, ³⁵ S-labeled and in vitro translated pontins were reacted in a hydrous mixture containing E1, E2 and ATP. (B) relates to a common point (ψKxE) search for fontin hydrophilization. ψ is an aliphatic amino acid and K is a lysine that conjugates to the hair. (C) Pontine's lysine 225 is the main condensation site. In vitro hydromyelination analysis was performed using wild type or K225R mutants of ³⁵ S-labeled and in vitro translated pontin as in (A) above. (D) 293T cells were simultaneously transfected with plasmids expressing Gal4-bound wild-type or K225R mutants of pontin in the presence of hair and Ubc9. Western blotting was performed using anti-Gal4 antibodies. (E) Immunoblot analysis shows the expression of flag-tagged pontin, hair-bound pontin, pontin K225R or hair-bound pontin K225R and shows schematic. (F) Shows subcellular localization in flag-tagged pontin, hair-bound pontin, pontin K225R, or hair-bound pontin K225R ponocytes in HeLa cells (green). The nucleus is shown via 4'-6-diamidino-2-phenylindole ( DAPI ) staining (blue).

Figure 3 shows that pontin hydration is required for the transcriptional activity of androgen receptor target genes. (A) When 5α-dihydroxytestosterone ( DHT ) is added in LNCaP cells, knockdown of pontin is associated with prostate-specific antigen ( PSA ), KLK2 , and NKX3 .1 translocation . Reduce carcass (B) When 5α-dihydroxytestosterone ( DHT ) was added to LNCaP cells, real-time RT-PCR analysis of shRNA treatment of pontin showed prostate-specific antigen ( PSA ). And KLK2 Transcript levels were down regulated. (C) KLK2, which is an androgen receptor target gene after introduction of pontin K225R or hairpin binding pontin K225R Transcript levels. It has been shown that the hydrophilization of (DF) pontin enhances the transcriptional function of pontin. Luciferase analysis was performed after DHT was added and cotransfection of the androgen responsive element ( ARE ) -luciferase reporter (D) . The expression of pontin activated the retinoid-related orphan receptor α2 element ( ROR α2E ) -luciferase reporter (E) , but the retinoic acid response element ( RARE ) -luciferase reporter (F) not activated. (G) Intracellular related experiments between pontin and nuclear receptors are shown. (H) Co-immunoprecipitation analysis to confirm the interaction of β-catenin, cAMP regulatory element-binding protein ( CBP ) or Tip60 with pontin or pontin K225R. shNS : non-specific shRNA, AR : androgen receptor, ROR α2 : Retinoid-related orphan receptor α2, RAR : retinoic acid receptor, RXR : retinoid X receptor, IP : immunoprecipitation, IB : immunoblotting, F- pontin : hairpin pontin

4 shows that pontin medification increases proliferation and growth of prostate cancer cells. (A) Hydrogenated pontin is increased in LNCaP cells when 5α-dihydroxytestosterone ( DHT ) is added. (B) Two-step chromatin immunoprecipitation ( ChIP ) of anti-pontin and anti-hairl IgGs resulted in the presence of a hair-modified pontin in the prostate-specific antigen ( PSA ) promoter under activation conditions. Indicates. (C) The results of ChIP analysis of Ubc9 on PSA promoters treated with DHT for 1 hour in LNCaP cells are shown. (D) shRNA for Ubc9 or non-specific shRNA (non-specific shRNA) the PSA androgen receptor target genes after introduction And KLK2 Represent the transcript. (E) Proliferation curves of mock-, pontin-K225R-, sumo-pontin- or sumo-pontin K225R-expressing LNCaP cells are shown. Values are expressed as mean ± sd of three independent experiments. (F) Adhesion-independent growth of pontin-, pontin-K225R-, sumo-pontin or sumo-pontin K225R-expressed LNCaP cells in soft agar medium. The image shown is an image of each group.

<110> SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION <120> Screening Methods of Anticancer Agent Using SUMOylation of Pontin <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1371 <212> DNA <213> Homo sapiens <400> 1 atgaagattg aggaggtgaa gagcactacg aagacgcagc gcatcgcctc ccacagccac 60 gtgaaagggc tggggctgga cgagagcggc ttggccaagc aggcggcctc agggcttgtg 120 ggccaggaga acgcgcgaga ggcatgtggc gtcatagtag aattaatcaa aagcaagaaa 180 atggctggaa gagctgtctt gttggcagga cctcctggaa ctggcaagac agctctggct 240 ctggctattg ctcaggagct gggtagtaag gtccccttct gcccaatggt ggggagtgaa 300 gtttactcaa ctgagatcaa gaagacagag gtgctgatgg agaacttccg cagggccatt 360 gggctgcgaa taaaggagac caaggaagtt tatgaaggtg aagtcacaga gctaactccg 420 tgtgagacag agaatcccat gggaggatat ggcaaaacca ttagccatgt gatcatagga 480 ctcaaaacag ccaaaggaac caaacagttg aaactggacc ccagcatttt tgaaagtttg 540 cagaaagagc gagtagaagc tggagatgtg atttacattg aagccaacag tggggccgtg 600 aagaggcagg gcaggtgtga tacctatgcc acagaattcg accttgaagc tgaagagtat 660 gtccccttgc caaaagggga tgtgcacaaa aagaaagaaa tcatccaaga tgtgaccttg 720 catgacttgg atgtggctaa tgcgcggccc caggggggac aagatatcct gtccatgatg 780 ggccagctaa tgaagccaaa gaagacagaa atcacagaca aacttcgagg ggagattaat 840 aaggtggtga acaagtacat cgaccagggc attgctgagc tggtcccggg tgtgctgttt 900 gttgatgagg tccacatgct ggacattgag tgcttcacct acctgcaccg cgccctggag 960 tcttctatcg ctcccatcgt catctttgca tccaaccgag gcaactgtgt catcagaggc 1020 actgaggaca tcacatcccc tcacggcatc cctcttgacc ttctggaccg agtgatgata 1080 atccggacca tgctgtatac tccacaggaa atgaaacaga tcattaaaat ccgtgcccag 1140 acggaaggaa tcaacatcag tgaggaggca ctgaaccacc tgggggagat tggcaccaag 1200 accacactga ggtactcagt gcagctgctg accccggcca acttgcttgc taaaatcaac 1260 gggaaggaca gcattgagaa agagcatgtc gaagagatca gtgaactttt ctatgatgcc 1320 aagtcctccg ccaaaatcct ggctgaccag caggataagt acatgaagtg a 1371 <210> 2 <211> 456 <212> PRT <213> Homo sapiens <400> 2 Met Lys Ile Glu Glu Val Lys Ser Thr Thr Lys Thr Gln Arg Ile Ala   1 5 10 15 Ser His Ser His Val Lys Gly Leu Gly Leu Asp Glu Ser Gly Leu Ala              20 25 30 Lys Gln Ala Ala Ser Gly Leu Val Gly Gln Glu Asn Ala Arg Glu Ala          35 40 45 Cys Gly Val Ile Val Glu Leu Ile Lys Ser Lys Lys Met Ala Gly Arg      50 55 60 Ala Val Leu Leu Ala Gly Pro Pro Gly Thr Gly Lys Thr Ala Leu Ala  65 70 75 80 Leu Ala Ile Ala Gln Glu Leu Gly Ser Lys Val Pro Phe Cys Pro Met                  85 90 95 Val Gly Ser Glu Val Tyr Ser Thr Glu Ile Lys Lys Thr Glu Val Leu             100 105 110 Met Glu Asn Phe Arg Arg Ala Ile Gly Leu Arg Ile Lys Glu Thr Lys         115 120 125 Glu Val Tyr Glu Gly Glu Val Thr Glu Leu Thr Pro Cys Glu Thr Glu     130 135 140 Asn Pro Met Gly Gly Tyr Gly Lys Thr Ile Ser His Val Ile Ile Gly 145 150 155 160 Leu Lys Thr Ala Lys Gly Thr Lys Gln Leu Lys Leu Asp Pro Ser Ile                 165 170 175 Phe Glu Ser Leu Gln Lys Glu Arg Val Glu Ala Gly Asp Val Ile Tyr             180 185 190 Ile Glu Ala Asn Ser Gly Ala Val Lys Arg Gln Gly Arg Cys Asp Thr         195 200 205 Tyr Ala Thr Glu Phe Asp Leu Glu Ala Glu Glu Tyr Val Pro Leu Pro     210 215 220 Lys Gly Asp Val His Lys Lys Lys Glu Ile Ile Gln Asp Val Thr Leu 225 230 235 240 His Asp Leu Asp Val Ala Asn Ala Arg Pro Gln Gly Gly Gln Asp Ile                 245 250 255 Leu Ser Met Met Gly Gln Leu Met Lys Pro Lys Lys Thr Glu Ile Thr             260 265 270 Asp Lys Leu Arg Gly Glu Ile Asn Lys Val Val Asn Lys Tyr Ile Asp         275 280 285 Gln Gly Ile Ala Glu Leu Val Pro Gly Val Leu Phe Val Asp Glu Val     290 295 300 His Met Leu Asp Ile Glu Cys Phe Thr Tyr Leu His Arg Ala Leu Glu 305 310 315 320 Ser Ser Ile Ala Pro Ile Val Ile Phe Ala Ser Asn Arg Gly Asn Cys                 325 330 335 Val Ile Arg Gly Thr Glu Asp Ile Thr Ser Pro His Gly Ile Pro Leu             340 345 350 Asp Leu Leu Asp Arg Val Met Ile Arle Thr Met Leu Tyr Thr Pro         355 360 365 Gln Glu Met Lys Gln Ile Ile Lys Ile Arg Ala Gln Thr Glu Gly Ile     370 375 380 Asn Ile Ser Glu Glu Ala Leu Asn His Leu Gly Glu Ile Gly Thr Lys 385 390 395 400 Thr Thr Leu Arg Tyr Ser Val Gln Leu Leu Thr Pro Ala Asn Leu Leu                 405 410 415 Ala Lys Ile Asn Gly Lys Asp Ser Ile Glu Lys Glu His Val Glu Glu             420 425 430 Ile Ser Glu Leu Phe Tyr Asp Ala Lys Ser Ser Ala Lys Ile Leu Ala         435 440 445 Asp Gln Gln Asp Lys Tyr Met Lys     450 455 <210> 3 <211> 306 <212> DNA <213> Homo sapiens <400> 3 atgtctgacc aggaggcaaa accttcaact gaggacttgg gggataagaa ggaaggtgaa 60 tatattaaac tcaaagtcat tggacaggat agcagtgaga ttcacttcaa agtgaaaatg 120 acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt tccaatgaat 180 tcactcaggt ttctctttga gggtcagaga attgctgata atcatactcc aaaagaactg 240 ggaatggagg aagaagatgt gattgaagtt tatcaggaac aaacgggggg tcattcaaca 300 gtttag 306 <210> 4 <211> 101 <212> PRT <213> Homo sapiens <400> 4 Met Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys   1 5 10 15 Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser              20 25 30 Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys          35 40 45 Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe      50 55 60 Leu Phe Glu Gly Gln Arg Ile Ala Asp Asn His Thr Pro Lys Glu Leu  65 70 75 80 Gly Met Glu Glu Glu Asp Val Ile Glu Val Tyr Gln Glu Gln Thr Gly                  85 90 95 Gly His Ser Thr Val             100  

Claims (16)

delete delete Culturing the cells; Contacting the cell with a potential substance; Measuring the hydrophilization of pontin in cells contacted with the potential material and control cells (cells not contacted with the potential material) using a promoter assay; And Comparing the measured degree of pontin with the degree of feminization, and selecting the latent substance that reduces the pontine with respect to the control cell, as an anticancer agent. The method of claim 3, wherein the promoter assay (promoter assay) characterized in that the expression level of the reporter functionally linked to the androgen receptor (androgen receptor) or Retinoid related orphan receptor ( ROR ) promoter Anticancer Screening Method. delete The method of claim 4, wherein the reporter is luciferase. 7. The method of claim 3, 4 or 6, wherein the cancer is prostate cancer. The method for screening an anticancer agent according to any one of claims 3, 4 or 6, wherein the cells are cancer cells. The method of claim 8, wherein the cancer cells are prostate cancer cells. 10. The method of claim 9, wherein the prostate cancer cells are LNCaP cells. Preparing a solution containing pontin and proteins necessary for hematuration (a hair activating enzyme, a hair binding enzyme, a hair ligase) and ATP; Treating the potential material with the solution; Measuring hydroponification of pontin in the solution and control (contact with the potential material) without contacting the potential material; And Comparing the measured degree of hydroponification of pontin, and selecting the potential substance as an anticancer agent that reduces the hydroponification of pontin in a solution in contact with the potential substance than in the control group. Way. 12. The method of claim 11, wherein the method for measuring pontin hydration is characterized by using an immunochemical method. 13. The method of claim 12, wherein the immunochemical method is measured by ELISA. 12. The method of claim 11, wherein the method for measuring hydroponification of the pontin is performed using radioisotope material. 12. The method of claim 11, wherein the method for measuring the hydroponification of pontin is based on the molecular weight difference according to electrophoresis. The method of claim 11, wherein the method for measuring the hydroponification of the pontin is anti-cancer screening method, characterized in that using a fluorescent dye.

Images