KR20170097370A - Biomarker composition for diagnosing bone metastatic breast cancer comprising IL-22 - Google Patents
Biomarker composition for diagnosing bone metastatic breast cancer comprising IL-22 Download PDFInfo
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Abstract
Description
The present invention relates to a biomarker composition for the diagnosis of osteosarcoma breast cancer comprising IL-22 as an active ingredient.
Interleukin-22 (IL-22) has 20% amino acid homology with IL-10 and is considered a member of the cytokine IL-10 family. IL-22 was originally referred to as an IL-10-related T-cell derived factor. IL-22 was mainly produced in active T helper 17 (Th17) cells, T helper 22 (Th22) cells and natural killer (NK) cells. The IL-22 receptor (IL-22R) belongs to the class II cytokine receptor family and is composed of two subunits, the specific IL-22R1 and the general IL-10R2. IL-22R1 is found in a variety of nonimmune tissues such as skin, lung, kidney and pancreas, while IL-10R2 is widely expressed in immune cells such as T, B and NK cells. IL-22 is a potent activator of STAT3. IL-22 binds to the IL-22R complex and induces Jak1 and Tyk2 signaling pathways including MAPKs (ERK, MEK, JNK and p38 kinase), STAT1, STAT3 and STAT5. IL-22 induces STAT3 phosphorylation mainly on tyrosine and serine residues and strongly activates the ERK1 / 2 pathway, whereas IL-10 signaling induces phosphorylation of STAT3 tyrosine residues.
Under inflammatory conditions, IL-22 levels in inflammatory sites increase, leading to the expression of proinflammatory molecules such as IL-1, IL-6, and IL-11. In addition, the elevated expression of IL-22 has been clinically reported in patients with inflammatory diseases including inflammatory bowel disease, rheumatoid arthritis (RA). Recent reports have suggested that IL-22 increases in synovial tissue and serum, and that up-regulation is associated with disease severity. Increased IL-22 promoted proliferation of synovial fibroblasts and induced CCL2 production, which resulted in synovium transfer of monocytes. Synovial fibroblasts secrete the nuclear factor kappa-B ligand (RANKL) in response to IL-22, leading to osteoclastogenesis.
Breast cancer is the most common cancer in women, and the five-year cure rate for non-metastatic breast cancer is about 99%. However, when recurred, cancer spreads to other organs, increasing the mortality rate. Breast cancer has a good metastatic spread to the bone. Metastasis to the bone tends to cause severe fractures, bone pain, hypercalcemia, and spinal cord compression syndrome. Skeletal complications due to bone metastasis severely affect the quality of life of patients. During bone metastasis, interaction with tumor cells and bone cells is essential. Bone metastases represent osteolytic, osteoblastic, or mixed lesions. Osteolytic lesions significantly increase osteoclast number and decrease osteogenic activity. Breast cancer cells produce a variety of factors that activate osteoclast precursor cells, such as IL-1, IL-6 and IL-11, which promote the production of RANKL in infiltrating T-cells, osteogenic cells and synovial fibroblasts . Secreted RANKL binds to the RANK receptor on the surface of osteoclast precursor cells, which leads to the formation of polynuclear osteoclasts with reabsorption activity. Unfortunately, bisphosphonate and denosumab, currently therapeutic agents for bone metastases, have limited efficacy in breast cancer patients with bone metastases. Therefore, it is necessary to understand the cell and molecular mechanisms involved in osteolysis and to find a new molecular therapeutic target in bone metastasis of breast cancer.
The present invention relates to a biomarker composition for the diagnosis of osteo-metastatic breast cancer comprising interleukin-22 (IL-22) as an active ingredient, a bone metastatic breast cancer comprising an agent capable of measuring the expression level of IL- A pharmaceutical composition for the prevention or treatment of osteo-metastatic breast cancer, which comprises an IL-22 protein expression or activity inhibitor as an active ingredient, and a method for screening a therapeutic agent for osteo-metastatic breast cancer through measurement of IL-22 protein expression level.
In order to solve the above problems, the present invention provides a biomarker composition for diagnosing osteosarcoma breast cancer, which comprises interleukin-22 (IL-22) as an active ingredient.
The present invention also provides a composition for the diagnosis of osteosarcoma breast cancer, which comprises, as an active ingredient, a preparation capable of measuring the expression level of IL-22.
The present invention also provides a method for providing information necessary for the diagnosis of osteosarcoma breast cancer by measuring the expression level of IL-22.
In addition, the present invention provides a pharmaceutical composition for preventing or treating osteo-metastatic breast cancer, which comprises an IL-22 protein expression or activity inhibitor as an active ingredient.
In addition, the present invention provides a method for screening a therapeutic agent for osteosarcoma breast cancer by measuring the expression level of IL-22 protein.
The present invention relates to a biomarker composition for the diagnosis of osteosarcoma breast cancer comprising IL-22 as an active ingredient, wherein IL-22 produces sphingosine-1-phosphate (S1P) And regulated breast cancer cell metastasis in a S1P-dependent manner. In other words, IL-22 plays an important role in the interaction between bone microenvironment and osteosarcoma breast cancer, which is likely to be used as a therapeutic target for breast cancer-related bone metastasis.
Figure 1 shows that IL-22 up-regulates S1P receptor expression in human breast cancer patients and bone metastatic human breast cancer cells. Results were expressed as mean and standard deviation. * P <0.05 compared with untreated cells.
Figure 2 shows that IL-22 increases S1PR1 mRNA and protein levels. Bars were expressed as the mean and standard deviation of triplicate experiments. * P <0.05 compared with untreated cells.
Figure 3 shows that IL-22 increases S1PR1 expression in MDA-MB 231 cells and increases cancer cell metastasis. Bars were expressed as the mean and standard deviation of triplicate experiments. * Compared with VPC24191 untreated cancer cells in the presence of P <0.05, IL-22 (10 ng / ml). # P <0.05, compared with VPC24191 treated cancer cells in the presence of IL-22 (10 ng / ml).
Thus, in order to confirm the molecular correlation between inflammation and breast cancer metastasis, the present inventors have found that the elevated levels of IL-22 affect the interaction between bone microenvironment, particularly osteoclasts and osteoblasts, and metastatic breast cancer under inflammatory conditions I found out. As a result, it was confirmed that IL-22 plays a potential role in promoting the metastatic potential of breast cancer cells and the bone metastasis-related mechanism of breast cancer, thus completing the present invention.
The present invention provides a biomarker composition for diagnosing osteosarcoma breast cancer comprising interleukin-22 (IL-22) as an active ingredient.
The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).
The present invention also provides a composition for the diagnosis of osteo-metastatic breast cancer, which comprises, as an active ingredient, a preparation capable of measuring the level of expression of interleukin-22 (IL-22).
Specifically, the agent capable of measuring the expression level of IL-22 may be a primer or a probe specifically binding to the IL-22 gene, an antibody specifically binding to the IL-22 protein, a peptide, Or a compound, but is not limited thereto.
The present invention also provides a kit for the diagnosis of osteo-metastatic breast cancer comprising the above composition.
As used herein, the term "primer" refers to a short nucleic acid that can form base pairs with a complementary template with a nucleic acid sequence having a short free 3 'hydroxyl group and serves as a starting point for template strand replication It refers to the sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.
As used herein, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to an mRNA. The presence or absence of a specific mRNA, You can check the amount. The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.
As used herein, the term "antibody" means a specific immunoglobulin as indicated in the art and directed against an antigenic site. An antibody in the present invention means an antibody that specifically binds to IL-22 of the present invention, and the antibody can be produced according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.
In addition, the kit of the present invention comprises an antibody specifically binding to a marker component, a secondary antibody conjugate conjugated with a label that develops upon reaction with the substrate, a coloring substrate solution to be colored with the label, A reaction stop solution, and the like, and may be manufactured from a number of separate packaging or compartments including the reagent components used.
As used herein, the term "peptide" has a high binding capacity to the target material and does not cause denaturation during thermal / chemical treatment. Also, because of its small size, it can be used as a fusion protein by attaching it to other proteins. It can be used as a diagnostic kit and a drug delivery material because it can be specifically attached to a polymer protein chain.
The term "aptamer " as used herein refers to a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid having a stable tertiary structure by itself and having a characteristic capable of binding with high affinity and specificity to a target molecule ). ≪ / RTI > As described above, since the aptamer is composed of a polynucleotide which is capable of specifically binding to an antigenic substance like the antibody and is more stable than the protein, has a simple structure, and is easy to synthesize, .
(1) measuring an IL-22 gene mRNA expression level or an IL-22 protein expression level from a sample isolated from a breast cancer patient; (2) comparing the mRNA expression level of the IL-22 gene or the expression level of the IL-22 protein with a control sample; And (3) determining that the bone metastatic potential is high when the level of mRNA expression of the IL-22 gene or the level of expression of the IL-22 protein is higher than that of the control sample. to provide.
In detail, the method for measuring the mRNA expression level may be RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) ), Northern blotting and DNA chips, but are not limited thereto.
In detail, the method for measuring the protein expression level may be Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, But are not limited to, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, Complement Fixation Assays, FACS and protein chips.
As used herein, the term "isolated sample from breast cancer patient" refers to a tissue, cell, whole blood, serum, plasma, serum, or plasma of a biomarker for the diagnosis of bone metastatic breast cancer, But are not limited to, saliva, sputum, cerebrospinal fluid, or urine.
In addition, the present invention provides a pharmaceutical composition for preventing or treating osteo-metastatic breast cancer, which comprises an IL-22 protein expression or activity inhibitor as an active ingredient.
Specifically, the IL-22 protein expression inhibitor may be an antisense nucleotide complementary to mRNA of the IL-22 gene, a small interfering RNA (siRNA) or a short hairpin RNA (shRNA) And the IL-22 protein activity inhibitor may be a compound, peptide, peptide mimetics, aptamer, antibody or natural product that specifically binds to IL-22 protein, but is not limited thereto.
Specifically, the IL-22 protein may induce expression of sphingosine-1-phosphate (S1P) receptor, but is not limited thereto.
The pharmaceutical composition of the present invention may contain a chemical substance, a nucleotide, an antisense, an siRNA oligonucleotide and a natural product extract as an active ingredient. The pharmaceutical composition or combination preparation of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a flavoring agent may be used. The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
Pharmaceutical dosage forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions . The pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI > The effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, once to several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg when administered once to several times a day, In the case of protein or antibody, 0.1 ng / kg to 10 g / kg, antisense nucleotide, siRNA, shRNAi or miRNA can be administered at a dose of 0.01 ng / kg to 10 g / kg.
(1) contacting a test substance to a breast cancer cell; (2) measuring the level of IL-22 protein expression or activity in a breast cancer cell in contact with the test substance; And (3) selecting a test substance having decreased expression or activity level of the IL-22 protein as compared with a control sample.
The term "test substance" used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening in order to examine whether it affects the expression amount of a gene or affects the expression or activity of a protein. do. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
< Experimental Example >
The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.
1. Reagents and antibodies
Recombinant mouse IL-22 protein, human tumor necrosis factor-alpha (TNF-alpha), mouse RANKL and human macrophage colony-stimulating factor (M-CSF) . (Rocky Hill, NJ). Leukocyte Acid Phosphatase Assay kit, PD98059, SB203580, SP60025, NS398 and β-actin antibodies were purchased from Sigma-Aldrich (St Louis, MO). Cell counting kit (CCK-8) was purchased from Dojindo (Kumamoto, Japan). RNAiMAX was purchased from Invitrogen (Grand Island, NY). Antibodies to p-ERK, ERK, p-p38, p38, p-JNK and JNK were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to p-STAT1, STAT1, p-STAT3, STAT3, p-STAT5 and STAT5 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Protease and phosphatase inhibitor cocktails were purchased from Thermo Pierce (Rockford, Ill.).
2. Cells Survival analysis
Cell viability was measured by CCK-8 colorimetric assay (Dojindo Molecular Technologies, Inc., Kunamoto, Japan). Briefly, cells were incubated for 1-6 days and then reacted in 10% CCK-8 solution at 37 ° C for 1 hour in culture medium. Optical density was measured at 450 nm on a microtitre plate reader (Bio-Rad, Hercules, Calif.).
3. Real time PCR And RT- PCR
RNA was isolated from the cells using QIAzol reagent (Qiagen, Hilden, Germany) and 1-2 μg of RNA was reverse transcribed with cDNA using the RevertiAid First-strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania). mRNA expression levels were analyzed by RT-PCR. Quantitative real-time PCR was performed using the Power SYBR Green 1-Step Kit and the ABI 7000 Real-Time PCR System (Applied Biosystems, Carlsbad, Calif.). The primer sequences used in the present invention are as follows: IL-22R, 5'-GAC TCC ATT TGG GTC ATC GC-3 '(SEQ ID NO: 1) and 5'- CCT GTC ACC GTG TGT CAT CA-3 '(antisense) (SEQ ID NO: 2); (SEQ ID NO: 3) and 5'-GAA GAG ACG TCC CAG AAC GG-3 '(antisense) (SEQ ID NO: 4); IL-10R, 5'-AAC GGA CAG GCA ATG ACG AA-3' (SEQ ID NO: 5) and 5'-AGC TTC TTC TCG CTC AGA CG-3 '(antisense) (SEQ ID NO: 6), IL-22, 5'-CAT GCA GGA GGT GGT ACC TT-3' ALP, 5'-CCA ACT CTT TTG TGC CAG AGA-3 '(sense) (SEQ ID NO: 7) and 5'-GGC TAC ATT GTT GAG CTT TT-3' (SEQ ID NO: 8); (SEQ ID NO: 9) and 5'-TGG TCT GAT AGC TCG TCA CAA G-3 '(SEQ ID NO: 10); Bglap2, 5'-CTG ACC TCA CAG ATC CCA AGC-3 (SEQ ID NO: 11) and 5'-GGA TGA GGA ATG CGC CCT A-3 '(antisense) (SEQ ID NO: 12); (SEQ ID NO: 13) and 5'-TCT ACC TGA GTG TCT TTG TTG ACT GTG-3 '(antisense) (SEQ ID NO: 14); COX2, 5'-TCA AAA GAA GTG CTG GAAAAG GTT-3' (SEQ ID NO: 15) and 5'-AAA GGT GCT GTA GGG GTT AG-3 '(antisense) (SEQ ID NO: 16); S1PR1, 5'-TCC ATG TAA ACT GGG TCA AG-3' S1PR2, 5'-TTT TAA AAT TGG GAC AGG GT-3 '(SEQ ID NO: 17) and 5'-TTC TCC ACA GGA TTT AGC AA-3' (SEQ ID NO: 18); (SEQ ID NO: 19) and 5'-TAT TTT TCC CTT AAC CCA GC-3 '(antisense) (SEQ ID NO: 20); S1'PR3, 5'-ATG GCA TTT GCT CTT GTT TA-3' (SEQ ID NO: 21) and 5'-ATA-CAG TTG GAA CAG TTG GG-3 '(SEQ ID NO: 22); S1'PR4, 5'-AAC TGT GGG TAT GAC TCT GG-3' EP4, 5'-ACC ATT CCT AGA TCG AAC CGT-3 '(sense) (SEQ ID NO: 23) and 5'-CAC CAC CCC GAA GAT GAA CAT-3' (SEQ ID NO: 24); M-CSF, 5'-GGC TTG GCT TGG GATGAT TCT-3 '(sense) (SEQ ID NO: 25) and 5'-GAG GGT CTG GCA ACT-3' (SEQ ID NO: 26); RANKL, 5'-AGC CGAGAC TAC GGC AAG TA-3 '(sense) (SEQ ID NO: 27) and 5'-AAA GTA CAG GAACAG AGC GAT G-3' (SEQ ID NO: 28); (OPG), 5'-CAG AGA AGC CACGCA AAA GTG-3 '(sense) (SEQ ID NO: 29) and 5'-AGC TGT GTC TCC GTT TTATCC T-3' (SEQ ID NO: 30); And GAPDH (glyceraldehyde 3-phosphatedhydrogenase), 5'-TGG CCT TCC GTG TTC CTA-3 '(sense) (SEQ ID NO: 31) and 5'- GAG TTG CTG TTG AAG TCG CA- ).
4. Enzyme-linked immunosorbent assay
Levels of soluble IL-22 in serum were measured by mouse IL-22 enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems). Briefly, recombinant mouse IL-22 serially diluted with assay diluent was used as a standard solution. Serum from the control, standard, wild-type and IL-22 knockout mice was reacted in three wells at room temperature for 2 hours. After washing with PBS containing 0.05
5. Small-Interference RNA Transfection
As a control, ON-TARGET plus non-targeting pool (D-001810-10-05) and ON-TARGET plus mouse IL-22R-specific small interfering RNA (siRNA) oligonucleotides (L- 057635-01-0005) Lafayette, CO). Pre-osteoblasts were transfected with siRNAs using transfection reagent RNAiMAX (Invitrogen, Carlsbad, Calif.).
6. Transwell Transition analysis
The MDA-MB 231 human breast cancer cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained according to the manual. Cells were cultured at 37 ° C in 5% CO 2 . MDA-MB 231 cells (5 × 10 4 ) pretreated with IL-22 for 24 hours were inoculated on top of an 8-μm pore size Transwell system (Costar, Corning, NY). Additives were added at the bottom. The Transwell assay was performed at 37 ° C for 6 hours. Cell migration through the inner membrane was quantitated by staining cells with hematoxylin. Cells on the top surface of the membrane were removed.
7. Immunity Blat analysis
Primary osteoblasts during the time displayed were treated with IL-22 (10 ng / ml ), cells naeteumyeo washed with cold PBS, protease and phosphatase containing the inhibitor Pro-pre TM Protein Extraction Solution (iNtRON Biotechnology, Seongnam , Korea). The cell lysate was centrifuged at 12,000 × g for 5 minutes, the supernatant was collected, and the protein was resolved in a 10% -12% SDS-PAGE gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.) And incubated in 5% skim milk dissolved in a tris-buffered saline solution containing 0.1% Tween for 1 hour Lt; / RTI > Membranes were reacted with appropriate primary antibodies overnight at 4 ° C, washed, and reacted with mustard peroperoxidase-conjugated secondary antibody at room temperature for 1 hour. The reaction protein was visualized using a chemiluminescence system (Merck-Millipore, Darmstadt, Germany).
< Example 1> in bone metastatic breast cancer cells S1P Lt; RTI ID = 0.0 > IL-22 < / RTI >
Expression of IL-22R1 is found in epithelium of skin, kidney, pancreas, liver and gastrointestinal tract, but not in immune cells. Conversely, IL-10R2 is expressed everywhere. Therefore, the present inventors confirmed whether breast cancer cells respond to IL-22. RT-PCR analysis showed that IL-22 significantly increased IL-22R1 mRNA expression in MDA-MB 231 human breast cancer cell lines compared to untreated control MDA-MB 231 cells (FIG. 1A). Although IL-22 is expressed in a variety of cells under inflammatory conditions, the role of IL-22 in breast cancer malignancy and metastasis is unclear. Because osteogenic S1P production promotes proliferation and metastasis of osteosaric prostate cancer, we have confirmed the expression of S1P receptor in MDA-MB 231 cells. To confirm that extrinsic IL-22 can affect the level of S1P receptor in MDA-MB 231 cells, the breast cancer cells were cultured with recombinant human IL-22 (10 ng / ml) and the S1PR1-S1PR5 mRNA levels were measured and RT-PCR was performed. S1PR1, S1PR2, S1PR3 and S1PR4 mRNA levels were increased in the IL-22-stimulated group compared to the untreated control group (Fig. 1B). However, IL-22 did not affect S1PR5 acting in neurons. The results indicate that IL-22 is able to modulate S1P receptor expression in metastatic breast cancer.
Breast cancer is often metastasized to the bone as well as essential organs such as the lungs, liver and brain. The present inventors analyzed the cohort of metastatic cancer patients using genome-wide gene expression data. S1P receptor expression analysis was performed between non-bone metastases (n = 47) and bone metastases (n = 17) using Gene Expression Omnibus (GEO) published data (GSE14020). Analysis showed that the expression of S1PR1, which exhibits a metastatic effect in cancer in bone metastasized cancer patients, is significantly increased (Fig. 2A). With attention to S1PR1, we performed Western blot analysis to confirm that extrinsic IL-22 affects its protein expression in MDA-MB 231 breast cancer cells. MDA-MB 231 cells were treated with IL-22 (10 ng / ml) for 72 hours. Using a density meter, it was confirmed that S1PR1 protein levels were significantly increased by IL-22 stimulation (FIG. 2B). Immunofluorescence analysis results were consistent with immunoblot results, and exogenous IL-22 increased S1PR1 expression (green) in a time-dependent manner (FIG. 2C). Nuclei were cross-stained with DAPI (blue). The results indicate that IL-22 increases mRNA and protein levels of S1PR1, which show a metastatic effect in metastatic breast cancer.
< Example 2> induced by IL-22 Increased S1PR1 Promoting breast cancer cell metastasis to bone microenvironment
According to the results of the present inventors, IL-22 has been shown to increase S1P production in osteoblasts. Thus, the present inventors confirmed that IL-22 affects the metastasis of metastatic breast cancer cells by increasing S1PR1 expression and S1P production in osteoblast cells in breast cancer cells. The present inventors used a Transwell transition analysis system. To improve breast cancer cell adhesion, the membrane was coated with 0.1% gelatin. MDA-MB 231 cells were treated or untreated with recombinant human IL-22 (10 ng / ml) for 24 hours and applied to Transwell transit assay. To preclude the effects of growth factors contained in fetal bovine serum, serum-free medium was used. MDA-MB 231 breast cancer cells (5 x 10 4 ) were pretreated with recombinant human IL-22 in serum-free medium for 24 hours, with a control (1% DMSO) or S1PR1 antagonist (VPC23019) at the top of the chamber. At the bottom of the chamber, a serum-free medium containing a control (1% DMSO) or a S1PR1 agonist (VPC24191), known as a chemotactic agent, was placed. The chamber was incubated for 6 hours, after which the cancer cells metastasized through the membrane were stained blue (FIG. 3A). Compared with the control, when the IL-22 and S1P receptor agonist were included, the number of metastatic breast cancer cells increased. When the S1P receptor antagonist was added, the effect of IL-22 on cell metastasis was blocked (FIG. 3B). That is, the results indicate that IL-22 increases metastasis through S1PR1 and promotes bone metastasis of breast cancer.
Having described the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> University of Ulsan Foundation for Industry Cooperation <120> Biomarker composition for diagnosing bone metastatic breast cancer comprising IL-22 <130> ADP-2015-0628 <160> 32 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gactccattt gggtcatcgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cctgtcaccg tgtgtcatca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aacggacagg caatgacgaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gaagagacgt cccagaacgg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 catgcaggag gtggtacctt 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 agcttcttct cgctcaga 18 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ccaactcttt tgtgccagag a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggctacattg ttgagctttt 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ctgacctcac agatcccaag c 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tggtctgata gctcgtcaca a 21 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ttcaacgatc tgagatctgt ggg 23 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggatgaggaa tgcgccct 18 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tcaaaagaag tgctggaaaa ggtt 24 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tctacctgag tgtctttgtt gactgtg 27 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tccatgtaaa ctgggtcaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 aaaggtgctg taggggttag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ttttaaaatt gggacagggt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ttctccacag gatttagcaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 atggcatttg ctcttgttta 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 tatttttccc ttaacccagc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aactgtgggt atgactctgg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 atacagttgg aacagttggg 20 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 accattccta gatcgaaccg t 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 caccaccccg aagatgaaca t 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ggcttggctt gggatgattc t 21 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gagggtctgg caact 15 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 agccgagact acggcaagta 20 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 aaagtacagg aacagagcga tg 22 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 cagagaagcc acgcaaaagt g 21 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 agctgtgtct ccgttttatc ct 22 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 tggccttccg tgttccta 18 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gagttgctgt tgaagtcgca 20
Claims (10)
(2) comparing the mRNA expression level of the IL-22 gene or the expression level of the IL-22 protein with a control sample; And
(3) determining that the bone metastatic potential is high when the mRNA expression level of the IL-22 gene or the IL-22 protein expression level is higher than that of the control sample.
(2) measuring the level of IL-22 protein expression or activity in a breast cancer cell in contact with the test substance; And
(3) A method for screening a therapeutic agent for osteo-metastatic breast cancer, comprising the step of selecting a test substance whose expression or activity level of the IL-22 protein is decreased as compared with a control sample.
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