KR101805181B1 - Use of Phosphorylated SLUG Protein for Novel Biomarker for Mitotic Cells - Google Patents

Abstract

The present invention relates to pSlug^(S158) protein, which is a Slug protein with the 158th serine residue phosphorylated, as a biomarker for mitotic cells. Since the pSlug^(S158) protein, which is a Slug protein with the 158th serine residue phosphorylated, is present specifically only in cells in mitosis, mitotic cells, in particular, cancer cells, can be effectively detected by using a binder that specifically binds to a phosphorylated site of the above protein. According to the present invention, the pSlug^(S158) protein may be beneficially used as a novel biomarker for mitotic cells including cancer cells.

Description

Use of Phosphorylated SLUG Protein as a Novel Biomarker for Mitotic Cells {Use of Phosphorylated SLUG Protein for Novel Biomarker for Mitotic Cells}

The present invention relates to the use of phosphorylated Slug protein as a novel biomarker for mitotic cells.

Slug protein is a transcriptional repressor belonging to the SNAIL family and plays an important role in the regulation of EMT (epithelial-mesenchymal transition). EMT is well known as a phenomenon that promotes cancer progression in cancer invasion and metastasis. Recently, Slug protein has been reported to be closely related to cell cycle as well as EMT. Slug protein-mediated cell cycle regulation is dependent on CDK2 (cyclin dependent kinase 2) and has been reported to modulate the G1 / S transition through the regulation of phosphorylation of S54 / S104 (Oncogene, 2015 Feb 26; 34 (9): 1116 -25).

There are anti-phospho-Histone H3S10 / S28 (PHH3) antibodies as an antibody used to detect conventional mitotic cells, but this PHH3 antibody has obvious limitations because it does not specifically react to mitotic cells.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Wang WL et al., Oncogene. 2015 Feb 26; 34 (9): 1116-25, Slug is temporally regulated by cyclin E in cell cycle and controls genome stability.

In studying the regulation mechanism of cancer by the Slug protein, the present inventors confirmed that the 158th serine (Serine 158, S158; Slug ^S158 ) residue of the Slug protein is phosphorylated at the miotosis stage during the cell division cycle , And the present inventors have completed the present invention by experimentally confirming that an antibody specifically binding to the phosphorylation site can highly specifically detect mitotic cells in cancer cells, various disease patients or tissues derived from cancer patients .

Accordingly, it is an object of the present invention to provide a biomarker of a mitotic cell such that the 158th serine residue of the Slug protein is phosphorylated (pSlug ^S158 ).

Another object of the present invention is to provide a kit for detecting mitotic cells comprising a binding agent that specifically binds to the phosphorylation site of the pSlug ^S158 protein as an active ingredient.

It is still another object of the present invention to provide a kit for the diagnosis or prognosis of cancer comprising as an active ingredient a binding agent that specifically binds to the phosphorylation site of the pSlug ^S158 protein.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a biomarker protein pSlug ^S158 of a mitotic cell in which the 158th serine residue of the Slug protein consisting of the amino acid sequence of SEQ ID NO: 1 is phosphorylated.

In the present invention, the Slug protein comprises the amino acid sequence of SEQ ID NO: 1.

The protein " pSlug ^S158 " in which the 158th serine residue of the Slug protein is phosphorylated appears specifically only in the mitotic phase of the cell cycle. The pSlug ^S158 can be used as a biomarker of mitotic cells.

According to another aspect of the present invention, there is provided a method for detecting mitosis, which comprises as an active ingredient, a binding agent that specifically binds to the phosphorylation site of the pSlug ^S158 protein, which is a phosphorylated form of the 158th serine residue of the Slug protein comprising the amino acid sequence of SEQ ID NO: A kit for detecting a cell is provided.

According to still another aspect of the present invention, there is provided a method for producing a pSlug ^S158 protein, which comprises a binding agent that specifically binds to the phosphorylation site of the pSlug ^S158 protein, which is a phosphorylated form of the 158th serine residue of the Slug protein comprising the amino acid sequence of SEQ ID NO: And provides a kit for diagnosing or prognosing cancer including cancer.

According to an embodiment of the present invention, the mitotic cell is a cancer cell. In this case, the kit of the present invention is a kit for cancer diagnosis or prognosis analysis. That is, the biomarker of the present invention can be used for the generation or diagnosis of cancer, and can be used for prognosis determination and analysis performed after cancer treatment.

As used herein, the term " diagnosis " is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of an object (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining a stage of a cancer, or determining the reactivity of a cancer to treatment), or determining therametrics And monitoring the status of the object to provide information about the object.

In the present invention, the cancer is preferably selected from the group consisting of pancreatic cancer, biliary cancer, neuroendocrine tumors, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, stomach cancer, bladder cancer, colon cancer, cervical cancer, But are not limited to, cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, pituitary cancer or urethral cancer.

According to one embodiment of the present invention, the phosphorylation site to which the binding agent specifically binds is the oligopeptide " CDAQSRK S FS " (SEQ ID NO: 2) region comprising the 158th serine in the Slug protein. The oligopeptide moiety is also referred to herein as CDAQSRK { pS ¹⁵⁸ } FS in the sense that the serine residue is phosphorylated.

According to one embodiment of the invention, the binding agent includes, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA) or an aptamer But is not limited thereto.

According to one embodiment of the present invention, the kit is an immunoassay kit and can be carried out by an immunoassay method, that is, an antigen-antibody reaction method. In this case, an antibody or an aptamer that specifically binds to the biomarker of mitotic cells is used.

The antibody used in the present invention is a polyclonal or monoclonal antibody. Antibodies can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), the recombinant DNA method (US Patent No. 4,816,56) Or phage antibody library methods (Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the method of the present invention is carried out using an antibody or an aptamer, the present invention can be carried out according to a conventional immunoassay method and used to diagnose cancer.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry,

But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e. G., C ^14, I ^125, P ³² and S ³⁵⁾ that the antibody is detected biomarkers danbaekjilreul of the present invention labeled with .

When the method of the present invention is carried out by an ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating the surface of a solid substrate with an unknown cell sample lysate to be analyzed; (Ii) reacting the cell lysate with an antibody to a marker as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as an enzyme that binds to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1 chromophore and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N (10-acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine ), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronin, glucose oxidase and nitroblue tetrazolium and m-PMS methosulfate) can be used. All.

When the method of the present invention is carried out in the Capture-ELISA mode, a specific embodiment of the present invention comprises the steps of (i) coating an antibody against the marker of the present invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the sample; (Iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating label and specifically reacts with an embedding protein; And (iv) measuring a signal originating from said label.

The detection antibody has a label that generates a detectable signal. Wherein the label is a chemical (e.g., biotin), an enzyme (alkaline phosphatase, β- galactosidase, horseradish peroxidase, and cytochrome P450), the radioactive material ((e.g., ¹⁴ C, ¹²⁵ I, P ³² and S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials and fluorescence resonance energy transfer (FRET). The various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. This detection of the signal enables a qualitative or quantitative analysis of the biomarker of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

According to another modification of the present invention, an aptamer that specifically binds to the biomarker of the present invention can be used instead of the antibody. Aptamers are oligonucleic acid or peptide molecules, and the general contents of aptamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

By analyzing the intensity of the final signal by the above-described immunoassay procedure, cancer can be diagnosed. That is, when the signal for the biomarker of the present invention is stronger than that of the normal sample in the sample, it is diagnosed as cancer.

According to another aspect of the present invention, there is provided a method for providing information necessary for the diagnosis or prognosis analysis of cancer, comprising the steps of: detecting a 158th serine residue of a Slug protein consisting of the amino acid sequence of SEQ ID NO: 1 in a biological sample separated from a human; Lt; RTI ID = 0.0 > pSlug < / RTI > ^S158 protein.

According to one embodiment of the present invention, the biological sample is human tissue, cell, blood, serum or plasma.

The present invention relates to the biomarker pSlug ^S158 protein of mitotic cells, which is a phosphorylated form of the 158th serine residue of the Slug protein. PSlug < / RTI > ^{S158 <} RTI ID = 0.0 > Proteins are specifically expressed only in cells undergoing mitosis. When a binding agent that specifically binds to the phosphorylation site of the protein is used, mitotic cells, particularly cancer cells, can be effectively detected. In the present invention, pSlug ^S158 Proteins can be usefully used as new biomarkers for mitotic cells containing cancer cells.

Figure 1 is an ELISA (Enzyme-Linked Immunosorbent Assay) result of anti-pSlug ^S158 antibody.
Fig. 2 shows the results of the anti-pSlug ^S158 antibody reacting to the tissue (panel A and panel B) generated in the PC-3 cell xenograft model and to the cancer cells PC-3 cells (panel C) and 293T cells (panel D) This is the result of confirming the reactivity.
FIG. 3 shows the tissue obtained from patients with various diseases and cancer diseases as anti (anti) -Slug ^S158 Antibody staining results are shown.
Panel A shows the tissue of patients with Kikuchi disease; Panel B shows the tissues of patients with cutaneous mastocytosis; Panel C shows the tissues of patients with basal cell carcinoma; Panel D shows the tissues of patients with esophageal squamous cell carcinomas; Panel E is the tissue of gastrointestinal stromal tumor (GIST) patients; Panel F is the tissue of a lymphoma patient; Panel G is the tissue of a patient with malignant melanoma; Panel G of FIG. 3), Panel H is tissue of leiomyosarcoma patient; Panel I is the result of staining tissues of patients with meningioma with anti-pSlug ^S158 antibody, respectively. It was observed that the cells react strongly in the nucleus of the mitotic cell.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Fabrication of antibodies capable of detecting phosphorylation at the 158th serine (S158) residue of the Slug protein

First, the following Slug peptide sequence [CDAQSRK { pS ¹⁵⁸ } FS] containing the phosphorylated 158th serine residue was used as an antigen. Peptide antigens were synthesized by Genescript USA Inc [860 Centennial Ave., Piscataway, NJ 08854, USA]. Antibodies were prepared using the above antigens. As a result of the antibody preparation, an antibody specifically reactive to the phosphorylated site of Slug ^S158 was obtained. The antibody titer was 1: 512,000 or more, and the result of ELISA using the antibody prepared is shown in Fig.

Example 2: Preparation of anti-pSlug ^S158 Immunohistochemical staining of cancer cells using antibodies

Experiments were conducted to confirm whether the antibody produced specifically reacted with cancer cells. For this, tumor tissues generated in a xenograft model prepared by transplanting PC-3 cells into a mouse using the antibody prepared in Example 1 were confirmed by immunohistochemistry (see panel A of FIG. 2). At this time, in order to confirm the specificity of the antibody, the test group in which the peptide used as the immunogen in the preparation of the antibody was added at a high concentration as a blocking peptide was used as a control group (see panel B in FIG. 2). In Panel A of FIG. 2, brown spots were found to have specifically reacted with the anti (anti) -Slug ^S158 antibody, whereas no brown spots were found in Panel B of FIG. 2, which is a control group to which the antigen peptide was added. As a result, it was confirmed that the brown dots of Panel A of FIG. 2 were the result of specific reaction with antibodies.

To further examine whether the antibody produced had cancer cell-specific reactivity, PC-3 cells and 293T cells of prostate cancer cells were treated with anti-pSlug ^S158 The antibodies were treated and reacted with secondary antibodies with fluorescence and observed under a fluorescence microscope (see Panel C and Panel D of FIG. 2). In both cells, anti-pSlug ^S158 antibody reacted at the nucleus of the mitotic cell (yellow arrow). This is an anti-pSlug ^S158 The antibody was found to be an antibody that reacts more strongly to the pSlug ^S158 protein, which is more specifically expressed in the nucleus of the mitotic cell.

Example  3: Anti - pSlug ^S158  Immunohistochemistry results of antibodies

A. Anti-pSlug targeting various tissues ^S158 Investigation of antibody reaction

Produced anti (anti) -pSlug ^S158 Immunostaining was performed on tissues obtained from patients with diseases using antibodies. Panel A of Figure 3 shows the tissue obtained from patients with Kikuchi disease as anti (anti) -Slug ^S158 This is the result of staining with antibody. It was observed that brown dots were strong in mitotic cells and not stained in apoptotic bodies, neutrophils and macrophages. This shows that the antibody of the present invention reacts very specifically to the nucleus of the cell where mitosis occurs. In addition, it can be observed that when the present antibody is stained in cutaneous mastocytosis, it is not stained at all in mast cells (see panel B of FIG. 3). This is a distinction from the anti-phospho-Histone H3S10 / S28 (PHH3) antibody, which is an antibody used as a typical indicator of mitosis.

B. An anti-pSlug ^S158  Immunostaining of antibodies

Panel C of FIG. 3 is the result of staining tissue of basal cell carcinoma patient with the antibody of the present invention. And the cytoplasm of cells in the basal layer of skin epithelium is weakly stained by anti-pSlug ^S158 antibody. No staining of this site has been reported when staining with PHH3 antibody. This is also the difference from the conventional mitotic indicator antibody, PHH3 antibody. In addition, it can be observed that the cells are stained strongly in the cells in the cancer tissue where the dividing cells are actively gathered.

Immunohistochemical staining of esophageal squamous cell carcinomas with anti-pSlug ^S158 antibody showed that the cells stained in nuclei and cytoplasm of metaphase cells of some cell division See panel D of FIG. 3). In particular, the nuclei were stained relatively strongly, whereas the cytoplasm was relatively weakly stained (insert image of panel D of FIG. 3).

Anti (anti) -Slug ^S158 Antibody-mediated cytoplasmic staining is observed only in squamous cell carcinomas, but not in epithelial tumors and mesenchymal tumors. pSlug ^S158 protein is specifically observed in the chromatin in the nucleus of the mitotic cell and is rarely observed in the nucleus of interphase cells during cell division. To confirm this, tissues from various cancer patients were immunostained with anti-pSlug ^S158 antibody. (GIST, panel E of FIG. 3), lymphoma (panel F of FIG. 3), malignant melanoma (panel G of FIG. 3), leiomyosarcoma H) and meningioma (panel I of FIG. 3) tissues were stained with an anti-pSlug ^S158 antibody. As a result, it was observed that the cells strongly reacted with nuclei of mitotic cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Use of Phosphorylated SLUG Protein for Novel Biomarker for          Mitotic Cells <130> PN150434 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 268 <212> PRT <213> Human Slug protein <400> 1 Met Pro Arg Ser Phe Leu Val Lys Lys His Phe Asn Ala Ser Lys Lys   1 5 10 15 Pro Asn Tyr Ser Glu Leu Asp Thr His Thr Val Ile Ile Ser Pro Tyr              20 25 30 Leu Tyr Glu Ser Tyr Ser Met Pro Val Ile Pro Gln Pro Glu Ile Leu          35 40 45 Ser Ser Gly Ala Tyr Ser Pro Ile Thr Val Trp Thr Thr Ala Ala Pro      50 55 60 Phe His Ala Gln Leu Pro Asn Gly Leu Ser Pro Leu Ser Gly Tyr Ser  65 70 75 80 Ser Ser Leu Gly Arg Val Ser Pro Pro Pro Ser Ser Asp Thr Ser Ser                  85 90 95 Lys Asp His Ser Gly Ser Glu Ser Pro Ile Ser Asp Glu Glu Glu Arg             100 105 110 Leu Gln Ser Lys Leu Ser Asp Pro His Ala Ile Glu Ala Glu Lys Phe         115 120 125 Gln Cys Asn Leu Cys Asn Lys Thr Tyr Ser Thr Phe Ser Gly Leu Ala     130 135 140 Lys His Lys Gln Leu His Cys Asp Ala Gln Ser Arg Lys Ser Phe Ser 145 150 155 160 Cys Lys Tyr Cys Asp Lys Glu Tyr Val Ser Leu Gly Ala Leu Lys Met                 165 170 175 His Ile Arg Thr His Thr Leu Pro Cys Val Cys Lys Ile Cys Gly Lys             180 185 190 Ala Phe Ser Arg Pro Trp Leu Leu Gln Gly His Ile Arg Thr His Thr         195 200 205 Gly Glu Lys Pro Phe Ser Cys Pro His Cys Asn Arg Ala Phe Ala Asp     210 215 220 Arg Ser Asn Leu Arg Ala His Leu Gln Thr His Ser Asp Val Lys Lys 225 230 235 240 Tyr Gln Cys Lys Asn Cys Ser Lys Thr Phe Ser Arg Met Ser Leu Leu                 245 250 255 His Lys His Glu Glu Ser Gly Cys Cys Val Ala His             260 265

Claims (11)

A biomarker pSlug ^S158 protein of the mitotic cell whose morphology is the phosphorylated 158th serine residue of the Slug protein consisting of the amino acid sequence of SEQ ID NO: 1.
A kit for detecting mitotic cells comprising as an active ingredient a binding agent that specifically binds to the phosphorylation site of the pSlug ^S158 protein, which is a phosphorylated form of the 158th serine residue of the Slug protein having the amino acid sequence of SEQ ID NO: 1.
The kit for detecting mitotic cells according to claim 2, wherein the phosphorylation site to which the binding agent specifically binds is CDAQSRK {pS ¹⁵⁸ } FS.
The method of claim 2, wherein the binding agent is an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer Kits for cell detection.
The kit for detecting mitotic cells according to claim 2, wherein the kit is an immunoassay kit.
A kit for the diagnosis or prognosis of cancer, comprising as an active ingredient, a binding agent that specifically binds to the phosphorylation site of the pSlug ^S158 protein, which is a phosphorylated form of the 158th serine residue of the Slug protein having the amino acid sequence of SEQ ID NO: 1.
The kit for diagnosing or prognosing cancer according to claim 6, wherein the phosphorylation site specifically binding to the binding agent is CDAQSRK { pS ¹⁵⁸ } FS.
The method of claim 6, wherein the binding agent is an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer. A kit for diagnostic or prognostic analysis.
The kit for diagnosing or prognosis of cancer according to claim 6, wherein the kit is an immunoassay kit.
A method for detecting pSlug ^S158 protein which is a phosphorylated form of the 158th serine residue of the Slug protein consisting of the amino acid sequence of SEQ ID NO: 1 in a biological sample isolated from a human is provided as a method for providing information necessary for diagnosis or prognosis analysis of cancer .
11. The method of claim 10, wherein the biological sample is human tissue, cell, blood, serum or plasma.

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