KR101634612B1 - AGR2 homo-dimer attenuating ER stress induced cell death - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer comprising an AGR2 (Anterior Gradient 2) dimer-specific inhibitor as an active ingredient. The present invention relates to a pharmaceutical composition for preventing and treating cancer, AGR2, which is overexpressed in metastatic tumors such as esophageal cancer, liver cancer, and lung cancer, is present in homo-dimer form through disulfide bonds in cancer cell lines, and AGR2 dimer weakens apoptosis caused by endoplasmic reticulum stress and interacts with BiP / GRP78, The AGR2 dimer specific inhibitor or antibody may be useful for diagnosing or treating cancer.

Description

AGR2 homo-dimer attenuating ER stress induced cell death

The present invention relates to a pharmaceutical composition for preventing and treating cancer containing a specific inhibitor of AGR2 dimer overexpressed in Hct8 human colon adenocarcinoma cell line as an active ingredient, and more specifically, specifically binding to AGR2 dimer. It relates to a cancer diagnostic kit containing an antibody and a method for screening cancer prevention or treatment.

The unfolded protein response (UPR) signaling mechanism is an intracellular stress response initiated by BiP/GRP78, a major regulator of intravesicular responses including protein biosynthesis, signal transduction, and calcium homeostasis (Palade, Science (Palade, Science ( 1975) 189:347-358; Ma and Hendershot, Cell (2001) 107:827-830; Zhang and Kaufman, J. Biol . Chem . (2004) 279:25935-25938; Clapham, Cell (2007) 131:1047 -1058; Dudek et al., Cell. Mol . Life Sci . (2009) 66:1556-1569). Dormant BiP/GRP78 is associated with endoplasmic reticulum stress transducers such as IRE1a (inositol-requiring kinase 1a), PERK (protein kinase RNA-like endoplasmic reticulum kinase) and ATF6 (activating transcription factor 6).

The mechanism of UPR signaling in response to endoplasmic reticulum stress is promoted by the dissociation of BiP/GRP78 from IRE1a and PERK, which is activated in the outer membrane of the endoplasmic reticulum by oligomerization and phosphorylation (Bertolotti et al., Nat. Cell Biol. (2000) 2 :326-332). In order to prevent additional translational loading and restore normal endoplasmic reticulum function, the activated UPR signaling mechanism weakens the translation of newly synthesized proteins. Moreover, the activity of UPR signaling enables the survival of tumor cells (Ma and Hendershot, Nat Rev. Cancer (2004) 4:966-977), and the reduction of BiP/GRP78 expression (knockdown) in mice inhibits tumor development. (Jamora et al., Proc. Natl. Acad. Sci. USA (1996) 93:7690-7694). In addition, XBP1 and PERK are also essential for tumorigenesis, and reduction of XBP1 expression in mice cannot cause metastatic tumors (Romero-Ramirez et al., Cancer Res. (2004) 64:5943-5947). Similarly, decreased PERK expression decreases the growth rate of tumors in vivo (Naczki et al., EMBO J. (2005) 24:3470-3481; Blais et al., Mol . Cell. Biol. (2006) 26:9517-9532).

Human Anterior Gradient 2 (AGR2) is homologous to XAG-2 of Xenopus laevis, which plays an essential role during neural development (Aberger et al., Mech. Dev. (1998) 72:115-130 ; Sive et al., Cell (1989) 58:171-180). In humans, AGR2 is present on an increased basis in most metastatic adenocarcinomas (e.g., breast cancer, pancreatic cancer, prostate cancer, gastric cancer, esophageal cancer, liver cancer, lung cancer, and colon cancer) (Brychtova et al., Cancer Lett. (2011) 304) :1-7). AGR2 is produced by various stresses such as hypoxia, serum depletion, and endoplasmic reticulum stress (Zweitzig et al., Mol . Cell. Biochem . (2007) 306:255-260; Zhao et al. , Dev . Biol . (2010) 338:270-279). The malignant tumor environment that induces stress due to reduced blood oxygenation promotes endoplasmic reticulum stress, and the response to the stress in solid cancer is linked to the developmental protein response signaling mechanism (Ma and Hendershot, Nat Rev. Cancer (2004)). ) 4:966-977; Bi et al., EMBO J. (2005) 24:3470-3481; Ron and Walter, Nat. Rev. Mol. Cell Biol. (2007) 8:519-529).

Overexpression of AGR2 promotes tumor growth and metastatic phenotype in vivo and in vitro (Wang et al., Cancer Res. (2008) 68:492-497), and also suppressing AGR2 expression (silencing) by UPR Although it is known to affect the signaling mechanism and autophagy due to endoplasmic reticulum stress (Higa et al., J. Biol. Chem. (2011) 286:44855-44868), AGR2 and the UPR signaling mechanism and the above are known to affect the autophagy. Although a number of reports have been reported on the association of cell death due to endoplasmic reticulum stress, studies on the molecular mechanisms for linking AGR2 and UPR signaling mechanisms are insufficient.

Accordingly, the present inventors have tried to confirm the molecular mechanism for the linkage of AGR2 and UPR signaling mechanisms, and as a result, AGR2 exists in the form of a dimer through disulfide bonds in cancer cell lines, and the AGR2 dimer is BiP/GRP78 and By confirming that cell death due to endoplasmic reticulum stress is suppressed through interaction, the present invention was completed by confirming that the specific inhibitor or antibody of the AGR2 dimer can be usefully used in cancer diagnosis and treatment.

An object of the present invention is to provide a pharmaceutical composition for preventing and treating cancer containing a specific inhibitor of AGR2 (Anterior Gradient 2) dimer as an active ingredient.

In addition, another object of the present invention is to provide a cancer diagnostic kit comprising an antibody that specifically binds to the AGR2 dimer.

In addition, another object of the present invention

1) treating the test compound to a cancer cell line as an experimental group;

2) measuring the amount or activity of the AGR2 dimer in the experimental group of step 1) and the control group not treated with the test compound, respectively; And

3) As a result of the measurement in step 2), it is to provide a method for screening cancer prevention or treatment comprising the step of selecting a test compound that decreases the amount or activity of the AGR2 dimer compared to the control group.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing and treating cancer containing a specific inhibitor of AGR2 (Anterior Gradient 2) dimer as an active ingredient.

In addition, the present invention provides a kit for diagnosis of cancer comprising an antibody that specifically binds to the AGR2 dimer.

In addition, the present invention

1) treating the test compound to a cancer cell line as an experimental group;

2) measuring the amount or activity of the AGR2 dimer in the experimental group of step 1) and the control group not treated with the test compound, respectively; And

3) As a result of the measurement in step 2), a method for screening cancer prevention or treatment is provided, comprising the step of selecting a test compound that decreases the amount or activity of the AGR2 dimer compared to the control group.

The present invention relates to a pharmaceutical composition for preventing and treating cancer containing a specific inhibitor of AGR2 (Anterior Gradient 2) dimer as an active ingredient, and includes colon adenocarcinoma, colon cancer, gastric cancer, breast cancer, pancreatic cancer, prostate cancer, AGR2, which is overexpressed in metastatic tumors such as esophageal cancer, liver cancer, and lung cancer, exists in a homo-dimer form through disulfide bonds in cancer cell lines, and the AGR2 dimer weakens cell death due to endoplasmic reticulum stress and interacts with BiP/GRP78, The specific inhibitor or antibody of the AGR2 dimer may be usefully used in cancer diagnosis or treatment.

1 is a diagram showing domains and motifs of AGR2 (Anterior Gradient 2) protein.
2 is a result of treatment with different concentrations of DSS, an intracellular chemical crosslinker, and BS3, an extracellular chemical crosslinker, showing the presence or absence of a monomer and a dimer of AGR2. It is a diagram showing.
Figure 3 is a FLAG tagged AGR2 wild-type and its CS-mutant, respectively, in Hct8 cells after cell-infected, 2-mercaptoethanol (2-mercaptoethanol) in the extract of the reduced cells and untreated cells tagged with FLAG This is a diagram confirming the resulting protein.
4 is a diagram showing the expression levels of FLAG, p-PERK, PERK, p-IRE1, IRE1, and tubulin in the extracted cells after cell infection with HeLa cells by varying the amount of AGR2 protein tagged with FLAG. to be.
FIG. 5 is an empty vector and a wild type of AGR2 tagged with FLAG and a mutant type thereof, respectively, after cell infection with HeLa cells, FLAG, p-PERK, PERK, p-IRE1, and the extracted cells as shown in FIG. 4. It is a diagram showing a comparison of the expression levels of IRE1 and tubulin.
6 is a statistical analysis of cell viability after treatment with tunicamycin (TM) and tapsigargin (TG) for 60 hours on the cell-infected cells as shown in FIG. 5 (*; p) It is a diagram showing the results of <0.05, **; p<0.005).
7 is a diagram illustrating the activity of caspase-3 after treatment with tunicamycin on the cell-infected cells as shown in FIG. 5.
Figure 8 is, in Hct8 cells each cell-infected with the AGR2 blank vector tagged with FLAG, after immunoprecipitation of AGR2 tagged with FLAG, the immunoprecipitated complex was subjected to Western blotting analysis with anti-BiP/GRP78 antibody to determine whether or not mutual binding. This is a confirmed degree.
9 is a diagram illustrating whether or not mutual binding was performed by immunoprecipitation of endogenous AGR2 in Hct8 cells treated with tunicamycin, and then immunoprecipitated complexes were subjected to Western blotting analysis with an anti-BiP/GRP78 antibody.
FIG. 10 is a diagram showing the results of co-transfected and immunoprecipitated HeLa cells in order to confirm the interaction of AGR2 wild-type tagged with FLAG and BiP/GRP78 tagged with HA.

Hereinafter, the terms of the present invention will be described in detail.

In the present invention, "improvement" refers to any action in which symptoms of cancer disease are improved or beneficially changed by administration of the composition.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. Can be administered. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing and treating cancer containing a specific inhibitor of AGR2 (Anterior Gradient 2) dimer as an active ingredient.

The AGR2 preferably has an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

The AGR2 dimer is preferably formed through a disulfide bond between cysteine, which is the 81st amino acid of the AGR2 monomer, but is not limited thereto.

The AGR2 dimer preferably inhibits cell death through interaction with BiP/GRP78, but is not limited thereto.

The AGR2 dimer-specific inhibitor preferably recognizes cysteine, which is the 81st amino acid of the AGR2 monomer in which a disulfide bond is formed, but is not limited thereto.

The cancer is preferably any one selected from the group consisting of colon adenocarcinoma, colon cancer, gastric cancer, breast cancer, pancreatic cancer, prostate cancer, esophageal cancer, liver cancer, and lung cancer, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors transfected using human colon adenocarcinoma Hct8 cell line and HeLa cell line to overexpress the AGR2 gene (see Fig. 1), and then confirmed the formation of AGR2 dimer through chemical crosslinking. (See Fig. 2).

In addition, it was confirmed that the AGR2 dimer inhibits cell death through the UPR signaling mechanism induced by endoplasmic reticulum stress upon AGR2 overexpression (see FIGS. 4 and 5). In addition, AGR2 dimer has resistance to endoplasmic reticulum stress and thus has high cell viability by weakening the activity of alpha-caspase-3, but when the 81st cysteine is mutated to serine, the AGR2 CS mutant is overexpressed and the AGR2 monomer is excessively present. It was confirmed that it did not significantly affect cell viability (see FIGS. 6 and 7 ).

In addition, as a result of immunoprecipitation with BiP/GRP78 and Immunoprecipitation for AGR2 wild type and AGR2 CS mutant type having almost the same expression level, the AGR2 wild type forms a dimer and directly interacts with BiP/GRP78, but the AGR2 CS mutation In the case of the older brother, it was confirmed that the interaction with BiP/GRP78 was weak (see FIG. 10).

Therefore, in the composition according to the present invention, AGR2 exists in the form of a homo-dimer through disulfide bonds in cancer cell lines, and the AGR2 dimer attenuates cell death due to endoplasmic reticulum stress and interacts with BiP/GRP78, so that the AGR2 dimer By containing a specific inhibitor or antibody as an active ingredient, it can be usefully used for cancer diagnosis or treatment.

The composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose , Mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , Sucrose, dextrose, sorbitol and talc, and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

That is, the composition of the present invention can be administered in various oral and parenteral dosage forms at the time of actual clinical administration.When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It can be prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in chaparral extract or a fraction thereof. It can be prepared by mixing sucrose, lactose, or gelatin. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The composition of the present invention may be administered by subcutaneous injection, intravenous injection, or intramuscular injection when administered parenterally.

Dosage units may contain, for example, 1, 2, 3 or 4 times or 1/2, 1/3 or 1/4 times the individual dosage. Individual dosages specifically contain the amount of the effective drug administered at one time, which is usually all, 1/2, 1/3 or 1/4 times the daily dosage. The effective dose of the composition of the present invention may be 0.0001 to 10 g/kg, specifically 0.001 to 1 g/kg, and may be administered 1 to 6 times a day. However, since it may increase or decrease according to the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.

In addition, the present invention provides a kit for diagnosis of cancer comprising an antibody that specifically binds to the AGR2 dimer.

Specifically, the AGR2 preferably has an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

The AGR2 dimer is preferably formed through a disulfide bond between cysteine, which is the 81st amino acid of the AGR2 monomer, but is not limited thereto.

The AGR2 dimer preferably inhibits cell death through interaction with BiP/GRP78, but is not limited thereto.

Therefore, in the cancer diagnostic kit according to the present invention, AGR2 is present in a homo-dimer form through a disulfide bond in a cancer cell line, and the AGR2 dimer attenuates cell death due to endoplasmic reticulum stress by interacting with BiP/GRP78. By including an antibody specific to the disulfide-binding portion of the dimer, it can be usefully used for cancer diagnosis.

The kit according to the present invention can be used to measure one or more markers of the present invention with differences in expression in cancer patients and normal persons. The kit of the present invention not only enables a medical practitioner such as a doctor to diagnose cancer by distinguishing whether a patient is cancer or not, but also monitors the patient's response to the treatment and makes it possible to change the treatment according to the result. . In addition, it can be used to identify compounds that regulate the expression of one or more markers in vivo or ex vivo in cancer models (eg, animal models such as mice and rats).

The cancer diagnostic kit of the present invention includes (i) a first acquisition reagent that binds to the 81st amino acid cysteine site of the discovered AGR2 monomer and (ii) a second acquisition reagent that does not bind to the first acquisition reagent. can do.

The first acquisition reagent is an antibody or metal chelate, preferably an antibody, and the second acquisition reagent is a secondary antibody with a conjugate labeled with a color developing enzyme, a fluorescent substance, a radioactive isotope or a colloid. The coloring enzyme may be peroxidase, alkaline phosphatase, or acid phosphatase (e.g. horseradish peroxidase), and in the case of a fluorescent substance, fluorescein carboxylic acid ( FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein-6-yl, dihydrotetramethylrosamin-4-yl, tetramethylrhodamine-5-yl, tetra Methylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl or 4,4-difluoro- 5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl is possible.

When a sample obtained from a patient is brought into contact with a first acquisition reagent, preferably an antibody that specifically binds to cysteine, which is the 81st amino acid of the AGR2 monomer with a disulfide bond, the sample can be diluted to an appropriate degree before contact with the antibody. The antibody may be immobilized on a solid phase to facilitate subsequent steps such as washing or separation of the complex. The solid form may be glass or plastic, for example a microtiter plate, a rod, a bead, or a microbead. Antibodies can also be bound to a probe substrate or protein chip. After the sample is placed at a constant temperature with the antibody, the antibody-marker complex can be measured by washing. This is carried out by placing the washed mixture at a constant temperature with a secondary acquisition reagent, preferably a secondary antibody. Measurement of the amount or detection of the presence of the antibody-marker complex may be performed through fluorescence, luminescence, chemiluminescence, absorbance, reflection, or transmission. In addition to the above method, the marker in the sample may be detected by performing an indirect analysis method, for example, a monoclonal antibody that binds to another epitope of the marker, and a competition or inhibition reaction analysis.

In addition, the kit may include a washing solution or an eluent capable of removing only the bound biomarker while removing the substrate for color development reaction with the enzyme and unbound protein. Samples used for analysis include biological samples capable of identifying disease-specific polypeptides that can be distinguished from normal conditions such as serum, urine, and tear saliva. Preferably, it can be measured from a biological liquid sample, for example blood, serum, plasma, more preferably, serum. Samples can be prepared to increase the detection sensitivity of the marker. For example, serum samples obtained from patients are anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, and continuous extraction. (sequential extraction) or gel electrophoresis may be used.

In addition, the present invention

1) treating the test compound to a cancer cell line as an experimental group;

*2) measuring the amount or activity of the AGR2 dimer in the experimental group of step 1) and the control group not treated with the test compound, respectively; And

3) As a result of the measurement in step 2), a method for screening cancer prevention or treatment is provided, comprising the step of selecting a test compound that decreases the amount or activity of the AGR2 dimer compared to the control group.

Specifically, the amount of the AGR2 dimer in step 2) is preferably measured using Western blotting, ELISA (Enzyme-linked immunosorbent assay), and immunoprecipitation, but is not limited thereto.

The cancer is preferably any one selected from the group consisting of colon adenocarcinoma, colon cancer, gastric cancer, breast cancer, pancreatic cancer, prostate cancer, esophageal cancer, liver cancer, and lung cancer, but is not limited thereto.

The screening method for cancer prevention or treatment according to the present invention is to select a test compound having a characteristic of inhibiting the formation of AGR2 dimer or dissociating a disulfide bond between AGR2, and the test compound selected by the above method inhibits AGR2 dimer. It can be usefully used for cancer prevention and treatment.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only to specifically illustrate the present invention, and the contents of the present invention are not limited thereto by the following examples.

<Example 1> Cell culture and endoplasmic reticulum (ER) stress treatment

DMEM (Dulbecco's Modified Eagle) containing 10% FBS (fetal bovine serum) for human colon adenocarcinoma (Hct8) cell line (Korea Cell Line Bank) and HeLa cell line (Korea Cell Line Bank) at 5% CO _{2 and 37°C.} Media) cultured in medium. After the cells were arranged at 0.5 to 1 x 10 ⁶ cells/well, 2 μg/mL tunicamycin (Sigma) and 0.25 μM tapsigargin (Sigma) were treated to induce endoplasmic reticulum stress. All experiments were repeated at least 3 to ensure reproducibility.

<Example 2> Plasmid preparation and transfection

AGR2 has a protein disulfide isomerase (PDI)-like domain composed of a CXXS domain, also called a Tx-like domain (Fig. 1). A full-length cDNA clone encoding human AGR2 (SEQ ID NO: 1) and BiP/GRP78 was purchased from Invitrogen, which was subcloned into a mammalian expression vector.

Specifically, the sequence coding for AGR2 and BiP/GRP78 is pCMV-Tag 1 (Agilent Technologies) without a tag using the BamHI/XhoI site and a FLAG (or HA) tag at the N-terminus. It was cloned into the containing pcDNA3.1 zeo (+) vector. The FLAG-AGR2 construct with the FLAG tag added to the entire AGR2 sequence was previously reported (Park et al., The protein disulfide isomerase AGR2 is essential for production of intestinal mucus, Proc . Natl . Acad . Sci. USA (2009) 106:6950-6955).

In addition, using a PCR-based site-directed mutagenesis method, pCMV-Tag 1 and pcDNA3.1 zeo (+) AGR2(C81S) mutations (cysteine, the 81st amino acid) were serine ) Substituted (CS) AGR2 mutant) constructs were prepared, respectively. Then, 1 μg of DNA plasmid was transfected into cells cultured in <Example 1>, respectively, using Lipofectamine 2000 reagent (Invitrogen).

Human AGR2 wild type amino acid sequence (SEQ ID NO: 1):

MEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEEALYKSKTSNKPLMIIHHLDE C PHSQALKKVFAENKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIMFVDPSLKADLLKADITALTGELKDNKADN

( C : 81st amino acid cysteine, a site where disulfide bonds between AGR2 monomers are formed)

<Example 3> AGR2 with a chemical cross-linking (Chemical crosslinking) Confirm dimer formation

According to our previous results (Lee do et al., Identification of proteins differentially expressed in gastric cancer cells with high metastatic potential for invasion to lymph nodes, Mol . Cells (2011) 31:563571), AGR2 is It is known to be associated with a high metastasis potential, and the AGR2 protein expressed in bacteria appears to be about 34 kDa in gel filtration chromatography, and this size indicates the dimer form. In addition, AGR2 forms a heterodimer with MUC2 through a disulfide bond using a cysteine 81 (Cys81) residue in mucus-producing cells.

Based on the above results, it was assumed that AGR2 may exist as a dimer in cells. To confirm this, after treating Hct8 cells with a chemical crosslinker, the cell extract was analyzed by Western blotting. Disuccinimidyl suberate (DSS), an intracellular chemical crosslinking agent, and bissulfosuccinimidyl suberate, an extracellular chemical crosslinking agent (BS ³ , Bis[sulfosuccinimidyl] suberate) are used. It was dissolved in DMSO or water immediately before use, respectively. Then, the cells were washed three times with ice-cold PBS and resuspended in 1 mL PBS, and then a chemical crosslinking agent was added to each cell suspension. After incubating the crosslinked cells at room temperature for 30 to 60 minutes, after the final concentration reached 15 mM, a quenching buffer solution (1 M Tris, pH 7.5) was treated at room temperature for 15 minutes to stop the chemical reaction. ^{DSS and BS 3} were treated with different concentrations of each cell suspension to 0, 0.1, 0.25, 0.5, 1.0, and 1.5 mM, respectively.

Specifically, in order to perform Western blotting, cells cultured for about 48 hours after cell infection according to <Example 2> were harvested and washed twice with ice cold PBS, and then the harvested cells were protease. NP-40 lysis buffer containing inhibitor and phosphatase inhibitor (Roche), composition: 20 mM TrisCl, pH 7.5; 137 mM NaCl; 1 mM EDTA; 1% NP-40; 10 % Glycerol). Separate cell lysates by centrifuging at 13,000 rpm for 20 minutes at 4°C. 1030 μg of protein in the supernatant was separated by SDS-PAGE, transferred to a PVDF membrane (Millipore), and then visualized using an antibody suitable as a probe and an ECL kit (Pierce). Tubulin was used as a control for all Western blotting experiments. Anti-AGR2 (Santa Cruz and Abcam) and anti-tubulin antibodies were used as primary antibodies.

As a result, as shown in FIG. 2, 17 and 34 kDa bands were identified, which were confirmed to be in the AGR2 monomer and dimer states, respectively (FIG. 2).

< Example 4> Confirmation of dimer formation through disulfide bonds between mutual molecules of AGR2

In order to investigate that the dimerization of AGR2 is a disulfide bond between mutual molecules, AGR2 wild-type and its CS mutant protein tagged with FLAG produced in Example 2 were overexpressed in Hct8 cells, and the cell extract was non-reducing. Western blotting was performed in the same manner as in <Example 3> using an anti-FLAG antibody (Sigma) in SDS-PAGE.

As a result, as shown in Figure 3, in the case of the non-reducing SDSPAGE gel, CS mutant AGR2 did not have a dimer form, but it was confirmed that the AGR2 wild type existed as a homo-dimer by intermolecular disulfide bonds ( Fig. 3).

< Example 5> AGR2 Confirmation of the effect of dimerization to inhibit endoplasmic reticulum stress-induced apoptosis

AGR2 is well known as a neoplastic and metastatic marker (Innes et al., Br. J. Cancer (2006) 94:1057-1065; Smirnov et al., Cancer Res. (2005) 65:4993-4997 ; Zhang et al., Genes Chromosomes Cancer (2005) 43:249-259; Liu et al., Cancer Res . (2005) 65:3796-3805; Chen et al., Mol . Cancer (2010) 9:149) , It is known that the increased expression level of AGR2 in tumor cells correlates with cell proliferation (Wang et al., Cancer Res . (2008) 68:492-497; Ramachandran et al., Cancer Res . (2008) 68 :7811-7818; Hrstka et al., Oncogene (2010) 29:4838-4847), AGR2 expression inhibition (silencing) influences the UPR mechanism (Higa et al., J. Biol . Chem. (2011) 286: 44855-44868) has been reported.

To confirm the effect of AGR2 overexpression on the UPR signaling mechanism based on the results of the prior literature, FLAG, p-PERK, PERK, p- when AGR2 was overexpressed in Hela cells of Example 1 above. Western blotting was performed in the same manner as in <Example 4> using an antibody for the expression levels of IRE1, IRE1, and tubulin. In the absence of endoplasmic reticulum stress, overexpression of AGR2 tagged with FLAG induced phosphorylation of PERK and IRE1a according to a dose-dependent manner (FIG. 4).

As a result, as shown in Fig. 4, after cell infection of 0, 0.25, 0.5, 1.0, and 2.0 μg of AGR2 proteins tagged with FLAG with HeLa cell lines, respectively, FLAG, p-PERK from the extracted cells through Western blotting , PERK, p-IRE1, IRE1, and the expression levels of tubulin were confirmed. At this time, anti-FLAG (Sigma), anti p-PERK (Santa Cruz), anti p-IRE1a (Abcam), anti BiP/GRP78, anti HA, anti PERK, anti IRE1a, anti PARP, or anti caspase -3 (Cell Signaling) antibody was used as the primary antibody.

* In addition, as shown in FIG. 5, in order to understand the function of AGR2 dimerization, AGR2 wild type or its CS mutant or empty vector tagged with FLAG was overexpressed by infecting cells with HeLa cells, respectively, and as a result, phosphorylated PERK and Although the amount of phosphorylated IRE1 increased, it was confirmed that the overexpressed CS mutant did not induce phosphorylation of either PERK or IRE1 (FIG. 5). Therefore, it was confirmed that the dimerization of AGR2 plays an important role in the activity of the UPR signaling mechanism.

<Example 6> Confirmation of cell viability of AGR2 dimerization

Cell Counting Kit-8 (CCK8, DOJINDO library) was used to determine cell viability under endoplasmic reticulum stress conditions.

Specifically, AGR2 wild type tagged with FLAG and its mutant or empty vector were transformed into HeLa cells cultured in <Example 2>, respectively, and then HeLa cells cultured in 96-well plates (5×10 ³ cells/ Well) was treated with tunicamycin or tapsigargin for 60 hours, then added to 10 μL of CCK8 assay solution, and the culture solution was further incubated for 1 hour. Then, absorbance was measured at 450 nm using an ELISA reader. Percent cell viability was calculated using the empty vector as 100%. All quantitative data were analyzed through an independent Student's t-test, and were judged to be significant when p<0.05.

As a result, as shown in Figure 6, the effect of siAGR2 (Higa et al., J. Biol . Chem. (2011) 286:44855-44868) and the transfected cells of the AGR2 wild-type overexpressed in the opposite to the endoplasmic reticulum stress. Although a result with resistance was shown, it was confirmed that the CS mutant overexpressing cells did not affect cell viability (FIG. 6).

<Example 7> Confirmation of α-caspase-3 activity of AGR2 dimerization

AGR2 wild type tagged with FLAG and its mutant or empty vector were transfected into the cultured HeLa cells of <Example 2>, and then treated with 2 μg/mL tunicamycin, and then α-caspase-3, Western blotting using antibodies against α- truncated caspase-3, Flag, and tubulin was performed to confirm the activity of α-caspase-3.

As a result, as shown in FIG. 7, the activity of α-caspase-3 (salt) exists due to prolonged endoplasmic reticulum stress, and α-cut caspase-3 is formed, and the cells are killed, but CS under endoplasmic reticulum stress Unlike mutant overexpression, it was confirmed that overexpression of AGR2 wild-type attenuates the activity of α-caspase-3 and inhibits α-truncated caspase-3 production. Therefore, it was confirmed from the above results that AGR2 dimerization attenuates cell death due to endoplasmic reticulum stress (FIG. 7).

<Example 8> Confirmation of the interaction of AGR2 dimer and BiP/GRP78

In order to investigate the possibility of interacting with AGR2 dimers and regulators of UPR signaling mechanisms such as BiP/GRP78, IRE1a, and PERK, exogenous FLAG expressed after cell infection in Hct8 cells according to Example 2 AGR2 tagged with was immunoprecipitation using FLAG M2 beads. Immunoprecipitation of AGR2 and BiP/GRP78 tagged with FLAG uses anti-FLAG M2 affinity gel (Sigma). To extract the protein tagged with FLAG, 500-600 μg of cell lysate with 20 μL of affinity gel was slowly rotated at 4° C. for 6 hours. The antigen-antibody complex obtained by centrifugation at 3000 rpm for 1 minute was washed 3 times with NP-40 lysis buffer, and then 1× SDS sample (sampling) buffer solution for 5 minutes to dissolve and separate the protein from the affinity gel. It was boiled at 95°C. Then, the immunoprecipitated complex was subjected to Western blotting using anti-BiP/GRP78, IRE1a, or PERK antibodies in the same manner as in <Example 4>.

As a result, as shown in FIG. 8, it was confirmed that BiP/GRP78, which serves as a central molecule chaperone in the process of UPR mechanism initiator and all endoplasmic reticulum, co-immunoprecipitation with AGR2, but PERK and IRE1a It was confirmed that AGR2 and co-immunoprecipitation did not occur (FIG. 8).

In addition, in order to determine whether there is a physical correlation between ARG2 and BiP/GRP78 under endoplasmic reticulum stress conditions, after immunoprecipitation of endogenous AGR2 with an anti-AGR2 antibody in Hct8 cells treated with 2 μg/mL tunicamycin, This immunoprecipitated complex was analyzed using an anti-BiP/GRP78 antibody.

As a result, the interaction of BiP/GRP78 and AGR2 increased in response to tunicamycin (FIG. 9).

In addition, in order to determine how dimerization of AGR2 affects the interaction with BiP/GRP78, AGR2 wild type or AGR2 CS-mutant type tagged with FLAG and BiP/GRP78 tagged with HA were used in the HeLa cell line. It was co-transfected using the transfection method of Example 2>, and co-immunoprecipitation was performed using HA-BiP/GRP78 complex and anti-HA agarose.

As a result, although the expression levels of AGR2 wild-type and AGR2 CS-mutant were almost the same, as the result of prior literature, AGR2 wild-type binds to BiP/GRP78, but the interaction between CS-mutant and BiP/GRP78 of AGR2 Was weak (Fig. 10).

<110> Korea Research Institute of Bioscience and Biotechnology <120> AGR2 homo-dimer attenuating ER stress induced cell death <130> 15p-12-045 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 175 <212> PRT <213> Homo sapiens <400> 1 Met Glu Lys Ile Pro Val Ser Ala Phe Leu Leu Leu Val Ala Leu Ser 1 5 10 15 Tyr Thr Leu Ala Arg Asp Thr Thr Val Lys Pro Gly Ala Lys Lys Asp 20 25 30 Thr Lys Asp Ser Arg Pro Lys Leu Pro Gln Thr Leu Ser Arg Gly Trp 35 40 45 Gly Asp Gln Leu Ile Trp Thr Gln Thr Tyr Glu Glu Ala Leu Tyr Lys 50 55 60 Ser Lys Thr Ser Asn Lys Pro Leu Met Ile Ile His His Leu Asp Glu 65 70 75 80 Cys Pro His Ser Gln Ala Leu Lys Lys Val Phe Ala Glu Asn Lys Glu 85 90 95 Ile Gln Lys Leu Ala Glu Gln Phe Val Leu Leu Asn Leu Val Tyr Glu 100 105 110 Thr Thr Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Val Pro Arg Ile 115 120 125 Met Phe Val Asp Pro Ser Leu Thr Val Arg Ala Asp Ile Thr Gly Arg 130 135 140 Tyr Ser Asn Arg Leu Tyr Ala Tyr Glu Pro Ala Asp Thr Ala Leu Leu 145 150 155 160 Leu Asp Asn Met Lys Lys Ala Leu Lys Leu Leu Lys Thr Glu Leu 165 170 175

Claims (6)

delete delete The amount of AGR2 (Anterior Gradient 2) dimer was measured in the samples taken from the experimental group suspected of having cancer, and the amount of AGR2 dimer was measured in the sample collected from the non-cancer control group. Wherein the amount of the dimer is greater than the amount of dimer of the control group.
4. The method according to claim 3, wherein the amount of the AGR2 dimer is measured using Western blotting, ELISA (enzyme-linked immunosorbent assay), or immunoprecipitation .
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