KR20200049384A - Composition for inhibiting stemness of stem cell comprising material for inhibiting phosphorylation of OCT4 and blocking activation of Wnt signal - Google Patents

Abstract

The present invention relates to a composition for inhibiting stemness of stem cells including a material for inhibiting phosphorylation of OCT4 and blocking Wnt signals. The material for inhibiting phosphorylation of OCT4 inhibits phosphorylation of the 351^th serine in mouse OCT4 or the 359^th serine in human OCT4 and inhibits Wnt signals, and thus can effectively reduce stemness of various stem cells. Therefore, the composition can be effectively used for inhibiting cancer proliferation, cancer recurrence, cancer metastasis, generation of resistance against anti-cancer agents in cancer, or the like. In addition, since the composition can reduce stemness in general stem cells, it is expected that the composition can reduce the time and can increase the efficiency in the differentiation of embryotic stem cells to specific cells.

Description

Composition for inhibiting stemness of stem cell comprising material for inhibiting phosphorylation of OCT4 and blocking activation of Wnt signal}

The present invention relates to a composition for suppressing the phosphorylation of OCT4 and the stem cell suppression of stem cells containing a substance that blocks the activation of the Wnt signal.

Most of the anti-cancer drugs that have been actively developed in recent years and used in anti-cancer treatment are drugs targeting rapidly proliferating cancer cells. In the case of anti-cancer treatment using these drugs, cancer cells are shown to be effectively killed at first, and cancer is cured, but cancer recurrence and / or metastasis may not occur because cancer stem cells remaining in the body are not removed. Recently, interest in cancer stem cells has recently increased because problems that indicate resistance to existing anticancer therapies frequently occur.

Cancer stem cells are cancer cells with unlimited regenerative capacity similar to normal stem cells. They proliferate slowly and differently from normal cancer cells. They are cancer cells with autogenous regeneration or differentiation ability that are unique to stem cells. It is known to have other mechanisms. Cancer stem cells show drug and radiation resistance in a specific subpopulation that exhibits the potential to differentiate into various cancer cell groups and self-renewal, which causes cancer recurrence as well as cancer initiation. Since it causes metastasis, preventing cancer stem cell proliferation is important in cancer treatment.

However, in the case of stem cell therapeutics that have been actively researched recently, it is difficult to control the proliferation, survival, etc. of stem cells, and thus side effects are caused. A typical side effect may be caused by the undifferentiated stem cells remaining in the body and becoming darkened after treatment is completed.

Therefore, if there is a method that can regulate the stem cell properties of stem cells, including cancer stem cells, it can be safely applied to various stem cell applications, and effectively suppress cancer recurrence, metastasis, and resistance by increasing the therapeutic effect of cancer. It is expected to be possible.

On the other hand, octamer-binding transcription factor 4 (OCT4), also known as POU5F1 (POU domain, class 5, transcription factor 1), is a transcription factor expressed in conventional embryonic stem cells and differentiation of cells It plays a role in preventing and disappears when natural differentiation of cells begins, and is known as a marker specific for embryonic stem cells that are starch-potentiating ability.

In addition, the Wnt signaling pathway has been identified as a potential target for cancer therapy. The Wnt signaling pathway is one of several important regulators of embryonic pattern formation, post-embryonic tissue maintenance, and stem cell biology. More specifically, Wnt signaling plays an important role in cell fate specification, including the development of cell polarity and self-renewal by stem cell populations. Unregulated activation of the Wnt pathway is associated with multiple human cancers, where it can change the developmental fate of these tumor cells so that the tumor cells remain in an undifferentiated proliferative state.

However, in the treatment of cancer, as previously known, OCT4 is not only used as a marker for confirming stem cellity, but further, OCT4 itself is directly regulated by regulating phosphorylation of a specific amino acid sequence among the amino acid sequences of OCT4, thereby stemming The link between OCT4 phosphorylation and stem cell regulation, such as regulating cellularity, is not known. In addition, the association between OCT4 phosphorylation and Wnt signal regulation and stem cell regulation, such as regulating Wnt signal with OCT4 phosphorylation regulation, has not been known.

Domestic Publication Patent 10-2014-0143989

The present invention is to solve the problems in the prior art as described above, by inhibiting the phosphorylation of OCT4 to suppress the stem cellity of stem cells and also includes a substance that blocks the Wnt signal as an active ingredient, cancer stem cells It is an object of the present invention to provide a composition capable of inhibiting stem cell properties of various stem cells.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the above object, the present invention comprises a substance that inhibits phosphorylation of serine at position 351 at the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or at position 359 at amino acid sequence of human OCT4. Provided is a composition for inhibiting stem cellity of cells.

In one embodiment of the present invention, the composition may further include a substance that blocks Wnt signal activation.

As another embodiment of the present invention, as an embodiment of the present invention, the amino acid sequence of the mouse OCT4 may be represented by the amino acid sequence of SEQ ID NO: 1.

As another embodiment of the present invention, the amino acid sequence of human OCT4 may be represented by the amino acid sequence of SEQ ID NO: 2.

As another embodiment of the present invention, the substance may include a substance that inhibits the expression of CSNK2A1.

As another embodiment of the present invention, the substance that inhibits the expression of CSNK2A1 may include short hairpin RNA (shRNA) represented by the nucleotide sequence of SEQ ID NO: 5 or 6.

As another embodiment of the present invention, the stem cells may be any one selected from the group consisting of embryonic stem cells, germ cells, and cancer stem cells.

As another embodiment of the present invention, the composition can inhibit the proliferation, self-renewal, colonization, or survival of stem cells.

In addition, the present invention comprises a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 and a Wnt signal activator, Provided is a kit for stem cell regulation.

In addition, the present invention comprises a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 and stem cells for cell therapy as an active ingredient. , Cell therapy.

As an embodiment of the present invention, the cell therapeutic agent may further include a substance that blocks Wnt signal activation.

In another embodiment of the present invention, the cell therapy may inhibit metastasis, survival, or cancer metastasis of therapeutic stem cells.

In addition, the present invention, in vitro , characterized in that it comprises the step of inhibiting phosphorylation of the 351-th serine in the amino acid sequence of human OCT4 or 359-th serine in the amino acid sequence of mouse OCT4 (octamer-binding transcription factor 4), It provides a method for suppressing stem cells through regulation of OCT4 phosphorylation function.

As an embodiment of the present invention, the method for inhibiting stem cells may further include blocking Wnt signal activation.

In addition, the present invention, in vitro , in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) inhibiting the phosphorylation of the 351th serine or the 359th serine in the amino acid sequence of human OCT4; And it characterized in that it comprises the step of activating the Wnt signal, provides a method for regulating stem cells through OCT4 phosphorylation.

In addition, the present invention comprises a pharmaceutical composition for preventing or treating cancer, comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 Gives

As an embodiment of the present invention, the pharmaceutical composition may further include an anti-cancer agent.

As another embodiment of the present invention, the pharmaceutical composition may inhibit cancer proliferation, survival, metastasis, relapse, or anticancer drug resistance.

As another embodiment of the present invention, the composition may further include a substance that blocks Wnt signal activation.

In addition, the present invention is an antibody for detecting whether the phosphorylation of the 359th serine in the amino acid sequence of the 351th serine or human OCT4 in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4), the amino acid sequence of SEQ ID NO: 7 or 8 It provides an antibody, which recognizes an epitope represented by and specifically binds to the 351th serine of phosphorylated mouse OCT4 or the 359th serine of phosphorylated human OCT4.

In addition, the present invention provides a method for screening a stem cell inhibitory substance of stem cells, comprising the following steps:

(a) processing a candidate substance in cells expressing mouse octamer-binding transcription factor 4 (OCT4) or human OCT4;

(b) determining whether phosphorylation of the 351th serine in the amino acid sequence of the mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4; And

(c) If the serine is not phosphorylated in step (b), selecting the candidate substance as a stem cell inhibitor of stem cells.

As an embodiment of the present invention, the screening method comprises the steps of (b), determining whether Wnt signal is activated in the cell; And

In the step (c), when the Wnt signal is not activated, it may include the step of selecting the candidate substance as a stem cell inhibitory substance of stem cells.

In addition, the present invention provides a method for providing information for diagnosing whether stem cells are inhibited by stem cells, comprising the following steps:

(a) determining whether phosphorylation of the 351 th serine in the amino acid sequence of mouse OCT4 or the 359 th serine in the amino acid sequence of human OCT4 from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4; And

(b) When the serine is not phosphorylated in the step (a), diagnosing that the stem cell properties of the stem cells are suppressed.

As an embodiment of the present invention, the method for providing information comprises the steps of (a), determining whether Wnt signal is activated from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4; And

In the step (b), when the Wnt signal is not activated, it may include a step of diagnosing that stem cellity of the stem cells is suppressed.

In addition, the present invention provides a method for preventing or treating cancer comprising administering the pharmaceutical composition to an individual.

In addition, the present invention provides a cancer prevention or treatment use of the pharmaceutical composition.

A substance that inhibits phosphorylation of OCT4 according to the present invention suppresses phosphorylation of 351th serine of mouse OCT4 or 359th serine of human OCT4, and thus can effectively reduce stem cell properties of various stem cells by suppressing Wnt signal. Therefore, it can be effectively used for suppressing the proliferation of cancer, suppressing recurrence of cancer, suppressing metastasis of cancer, and suppressing the occurrence of anticancer drug resistance of cancer, and can also reduce stem cellity in normal stem cells, so as specific cells of embryonic stem cells. In the differentiation of, time can be shortened and efficiency can be increased. In addition, when the OCT4 phosphorylation and Wnt signal activation inhibitory substance of the present invention is used for cell therapy using embryonic stem cells, undifferentiated embryonic stem cells remaining after treatment can be completely removed and effectively suppress side effects of cancer. It is expected that the stability of cell therapy using embryonic stem cells can be significantly increased.

1 is a diagram showing the sequence of 14 amino acids including the position 351 serine for the production of OCT4 phosphorylation specific antibody according to an embodiment of the present invention.
2 is a view showing the results of verification by recognizing the peptide of the corresponding phosphorylation position as an antibody during Western blotting for verification of the OCT4 phosphorylated antibody according to an embodiment of the present invention.
3 is a view showing the results of verifying whether OCT4 phosphorylation is recognized when inhibiting resistant OCT4 expression by treating doxycycline in an embryonic stem cell line according to an embodiment of the present invention.
4 is a view showing the results of verifying a phosphorylated antibody using an immunofluorescence staining method in an embryonic stem cell line according to an embodiment of the present invention.
5 is a view showing the results of verifying the OCT4 phosphorylated antibody according to an embodiment of the present invention through an immunoprecipitation method.
6 is a view showing the results confirming whether OCT4 phosphorylation is recognized in human cancer cells according to an embodiment of the present invention.
7 is a view showing the results confirming that the mutant OCT4 was expressed in a dose similar to that of the wild type (WT) in the transformed cell line according to an embodiment of the present invention.
8 is an alkaline phosphatase staining effect of the phosphorylation impossibility (S351A) and mimic mutation (S351E) of the 351 th serine of OCT4 in the transformed cell line according to an embodiment of the present invention on stem cell self-renewal ability. This is a diagram showing the results confirmed through the formation of the used embryonic stem cells.
9 is a view showing the results of confirming the transgenic embryonic stem cell line according to an embodiment of the present invention using Western blotting.
10 is a view showing the results of confirming the stem cell self-renewal ability of the transduced embryonic stem cell line according to an embodiment of the present invention using an alkaline phosphatase staining method.
11 is a view showing the results of confirming the stem cell self-renewal ability of the transduced embryonic stem cell line according to an embodiment of the present invention as a difference in SSEA-1 expression.
12 is a diagram showing the results of analyzing gene expression patterns and ChIP data of a transgenic embryonic stem cell line according to an embodiment of the present invention.
FIG. 13 is a diagram showing the results obtained by visualizing a path analysis result of FIG. 12 according to an embodiment of the present invention.
14 is a diagram showing the results of a gene set amplification analysis for a Wnt signal among the results of the pathway analysis of FIG. 13 according to an embodiment of the present invention.
15 is a view showing the results of confirming the stem cell self-renewal ability using an alkaline phosphatase staining method when artificially activating Wnt signal in a transduced embryonic stem cell line according to an embodiment of the present invention.
16 is a view showing the results of confirming the stem cell self-renewal ability by SSEA-1 expression when artificially activating Wnt signal in the transduced embryonic stem cell line according to an embodiment of the present invention.
17 is a view showing the results confirming that the kinase inducing phosphorylation of OCT4 according to an embodiment of the present invention is CSNK2A1.
18 is a view showing that CSNK2A1 (casein kinase 2 alpha 1) induces phosphorylation of OCT4 as an in vitro kinase according to an embodiment of the present invention.

The present invention provides a composition for inhibiting stem cell stem cells, comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4.

In the present invention, the composition may further include a substance that blocks Wnt signal activation.

In addition, the present invention comprises a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 and a Wnt signal activator, Provided is a kit for stem cell regulation.

At this time, the kit including the OCT4 phosphorylation inhibitory substance and the Wnt signal activator can suppress stem cell activity by inhibiting Wnt signal activation by inhibiting phosphorylation of OCT4 by the OCT4 phosphorylation inhibitory substance, and inhibiting the OCT4 phosphorylation Stem cellity of stem cells can be regulated by increasing the stem cellity again by inhibiting only the Wnt signal by the Wnt signal activator in the cells. For the above-mentioned stem cell regulation, the OCT4 phosphorylation inhibitory substance and the Wnt signal activating substance can act without limitation at least once, respectively, and there is no limitation after and after applying each substance, and application of each substance can be simultaneously performed. It could be micro or it could go on.

In the present invention, the kit is a container; directions; A substance that inhibits phosphorylation of serine 351 at the amino acid sequence of mouse OCT4 or 359 at the amino acid sequence of human OCT4; And a Wnt signal activating material. The container comprises a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of the mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4; And Wnt signal activating materials, respectively, and may serve to store and fix. The material of the container may be, for example, plastic, glass bottle, but is not limited thereto. In addition, the instructions included in the stem cell regulation kit for stem cells according to the present invention may include instructions for adjusting the stem cell characteristics of the stem cells, in a separate sheet or booklet from the container. Instructions may be written or the sheet or booklet may be included in the container together with the OCT4 phosphorylation inhibitory substance and Wnt signal activating substance.

In the present invention, the amino acid sequence of the mouse OCT4 can be represented by the amino acid sequence of SEQ ID NO: 1 (NCBI Reference Sequence: NP_038661.2), and the amino acid sequence of the human OCT4 is the amino acid sequence of SEQ ID NO: 2 (NCBI Reference Sequence) : NP_002692.2).

In the present invention, the amino acid sequence of SEQ ID NO: 1 may be encoded by the base sequence of SEQ ID NO: 3 (NCBI Reference Sequence: NM_013633.3).

In the present invention, the amino acid sequence of SEQ ID NO: 2 may be encoded by the base sequence of SEQ ID NO: 4 (NCBI Reference Sequence: NM_002701.5).

In the present invention, the substance may be any one selected from the group consisting of peptides, proteins, nucleotides, and compounds, preferably a substance that inhibits expression of CSNK2A1, and a substance that inhibits expression of CSNK2A1 is a sequence It may include a short hairpin RNA (shRNA) represented by the nucleotide sequence of No. 5 or 6, but inhibit phosphorylation of the 351th serine in the amino acid sequence of mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4. If possible, the material is not limited thereto.

The term "OCT4 (octamer-binding transcription factor 4)" in the present invention is expressed in pluripotent embryonic stem cells and germ cells as one of the family of POU transcription factors. Members of the POU transcription factor family have conserved DNA binding domains, or POU domains, which were first identified in the transcription factors Pit-1, Oct-1, Oct-2 and Unc-86. OCT4 activates transcription through an octamer motif located adjacent to or at a distance from the transcription initiation site.

In the present invention, the term "Wnt" is a secretory glycoprotein, a ligand for the transmembrane receptor Frizzled 7 times and a transmembrane receptor transmembrane low density lipoprotein receptor related protein 5,6 (LRP5 / 6). The Wnt gene family encodes a large group of secreted proteins associated with the Int1 / Wnt1 proto-oncogene and the Drosophila wingless ("Wg") homolog of Drosophila Wnt1. Wnt is expressed in various tissues and organs, and segmentation in Drosophila; Endoderm development in C. elegans; And the development of limb polarity in mammals, neuronal differentiation, renal morphogenesis, gender determination and brain development. The Wnt signal transduction pathway transcends species from nematodes, fruit flies to mammals, and is a major regulator of animal development during embryogenesis and in mature organisms.

In the present invention, the term "stem cell (stem cell)" refers to the broad concept of the ability to differentiate into various types of tissue cells, ie, undifferentiated cells with stem cellity (stemness). These stem cells are largely divided into embryonic stem cells, adult stem cells, adult stem cells, gamete, cancer stem cells, etc., which can be prepared using embryos. Stem cells refer to the stage of cell lumps before forming a specific organ within 14 days after fertilization, and recently, embryonic stem cells are also produced from normal cells through dedifferentiation. Therefore, the cells that can differentiate into all cells and tissues constituting the body are not limited thereto. Adult stem cells are extracted from umbilical cord blood, bone marrow, and blood, and mean primitive cells immediately before being differentiated into cells of specific organs such as bone, liver, and blood. Germ cells are cells that transmit genetic information to the next generation through reproduction, and humans have sperm and eggs, but are not limited thereto. In addition, the stem cells can self-replicate in the process of forming a cluster of cells by forming a clone, thereby maintaining one new stem cell in the cluster, and have the ability to form one or more characteristic cell types through differentiation. Cells.

In the present invention, the term "stemness" refers to the unique ability of stem cells, as well as pluripotency, which can differentiate into all kinds of cells constituting organisms such as nerves, blood, and cartilage. In addition, it includes both multipotency and unipotency, and includes both self-renewal and self-renewal. In addition, it means that it has the ability to form a cluster through self-replication or self-regeneration ability. At this time, the stem cellity may include the ability to differentiate embryonic stem cells obtained from embryos generated from fertilized eggs into various types of cells, and although functionally different, they are retained in each body tissue of an adult body. The ability to differentiate adult stem cells into different types of cells may also be included.

Cancer stem cells refer to cancer cells in a comprehensive sense having stem cell-specific self-renewal or differentiation ability, which is a characteristic of stem cells. Cancer stem cells generally proliferate at a slower rate than normal cancer cells under normal tumor growth conditions (referring to a state in which there is sufficient nutrients (glucose) required for cell growth and no growth of the tumor microenvironment, and no cell stress). Maintaining a dormant state may have resistance to anti-cancer agents. For example, the expression of transcription regulators such as PGC-1α is controlled unlike normal tumor cells, so that the function of major metabolic regulators is normal cancer cells. It may be different compared to. It refers to cells with invasion and metastasis that have acquired resistance to apoptosis in a nutritional deficiency state through the regulation of these different metabolic regulation capabilities and the cell signaling system mechanically linked thereto. However, it is not limited to cells that can differentiate into normal cancer cells.

In the present invention, the stem cells may be any one selected from the group consisting of embryonic stem cells, germ cells, and cancer stem cells, but the type of stem cells in which OCT4 protein is expressed is not limited thereto.

In the present invention, the composition can suppress stem cell proliferation of stem cells, thereby suppressing the proliferation, survival, self-renewal, and clustering ability of stem cells, thereby controlling stem cells in all fields using stem cells. It can be used in any way.

In addition, the present invention comprises a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 and stem cells for cell therapy as an active ingredient. , Cell therapy.

In the present invention, the cell therapeutic agent may further include a substance that blocks Wnt signal activation.

In the present invention, the term "cell therapy" refers to the proliferation or selection of autologous, allogenic, xenogenic cells in vitro to restore the function of cells and tissues, or changes the biological properties of cells by other methods. Drugs used for the purpose of treatment, diagnosis, and prevention through a series of methods such as prescribing.

In the present invention, the cell therapy may control the stem cells by inhibiting the metastasis and survival of the therapeutic stem cells, or may suppress the occurrence of cancer due to the cancer of the remaining therapeutic stem cells after treatment. If the effect is induced by suppressing the function is not limited to this.

In addition, the present invention, in vitro , characterized in that it comprises the step of inhibiting phosphorylation of the 351-th serine in the amino acid sequence of human OCT4 or 359-th serine in the amino acid sequence of mouse OCT4 (octamer-binding transcription factor 4), It provides a method for suppressing stem cells through regulation of OCT4 phosphorylation function.

In the present invention, the method for inhibiting stem cells may further include blocking Wnt signal activation.

In addition, the present invention, in vitro , in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) inhibiting the phosphorylation of the 351th serine or the 359th serine in the amino acid sequence of human OCT4; And it characterized in that it comprises the step of activating the Wnt signal, provides a method for regulating stem cells through OCT4 phosphorylation.

In addition, the present invention comprises a pharmaceutical composition for preventing or treating cancer, comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 Gives

In addition, the present invention provides a method for preventing or treating cancer comprising administering the pharmaceutical composition to an individual.

In addition, the present invention provides a cancer prevention or treatment use of the pharmaceutical composition.

The term "cancer" in the present invention is an aggressive property in which cells divide and grow, ignoring normal growth limits, an invasive property that penetrates surrounding tissue, and metastatic that spreads to other parts of the body. It is a generic term for diseases caused by cells with characteristics.

The main cause of cancer death is cancer metastasis and treatment resistance, and the fact that the root cause is stem cell cancer cells has been proved in recent years. Therefore, it is expected that cancer-related death can be substantially reduced through an anticancer strategy that reduces the stem cellity of cancer cells.

The path for cancer cells to acquire stem cellity can be broadly summarized into two types, one in which normal stem cells become cancerous, and the other in cases where cancer cells acquire stem cellity through dedifferentiation. . In cancer patients, in particular, the key factors of embryonic stem cells play an important role in the maintenance of stem cells of cancer cells, and as malignant cancer progresses, the expression of embryonic stem cell-related gene groups rather than normal tissue stem cell-related genes is reported to increase. Became.

The term "phosphorylation" in the present invention is a biochemical reaction that adds a phosphate group to a Ser (serine), Thr (threonine) or Tyr (tyrosine) residue of a specific protein, which is catalyzed by a protein kinase enzyme. Phosphorylation typically modulates the function of the target protein, thereby modulating the activity of the protein. As part of the cellular homeostatic mechanism, phosphorylation is only a temporary process, which is reversed by other enzymes called phosphatases. Any abnormality in any aspect of the reaction (phosphorylation vs. dephosphorylation) can disrupt cell function.

In another embodiment of the present invention, an antibody that specifically recognizes phosphorylation of the 351th serine of OCT4 was produced (see Example 1-1), and phosphorylation of the 351th serine of mouse OCT4 is human cancer cells (NCCIT and NTERA2). It was confirmed that it was also detected (see Example 1-2).

In another embodiment of the present invention, as a result of confirming the association between OCT4 phosphorylation and OCT4 activity, in the case of the OCT4 non-phosphorylated mutant cell line (S351A), the number of embryonic stem cells forming colonies decreases when compared to the wild type, whereas OCT4 The phosphorylation-mimicking mutant cell line of (S351E) confirmed that the number of colonies increased compared to the wild type, and thus, when phosphorylation of mouse 351 th serine (359 th serine of human OCT4) was inhibited, it was confirmed that stem cellity was reduced ( See Example 1-3), the substance that inhibits phosphorylation of the 351th serine of mouse OCT4 or the 359th serine of human OCT4 inhibits the function of OCT4, thereby causing normal stem cells to lose and differentiate stem cells. , Cancer cells lose stem cellity, making long-term survival impossible, and relapse after chemotherapy.

In another embodiment of the present invention, as a result of confirming the association between phosphorylation of OCT4 and Wnt signal regulation, when the non-phosphorylated mutant (S351A) OCT4 was expressed, it was confirmed that the Wnt signal was suppressed, and cells stably expressing the non-phosphorylated mutant OCT4 When Wnt signal was artificially activated, it was confirmed that the stem cell self-renewal ability was restored (see Example 2).

In another embodiment of the present invention, as a result of confirming the kinase inducing phosphorylation of the 351 th serine of OCT4, it was confirmed that CSNK2A1 induces phosphorylation of the 351 th serine of OCT4 (see Example 3).

In the present invention, the cancer is preferably made of thyroid cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, rectal cancer, colon cancer, blood cancer, etc. It may be, more preferably gastric cancer, colon cancer, lung cancer, liver cancer, prostate cancer, thyroid cancer, breast cancer, cervical cancer, ovarian cancer, etc., but is not limited to cancer associated with OCT4.

In the present invention, the term "prevention" means any action that suppresses or delays the onset of diseases such as cancer by administration of the composition according to the present invention.

In the present invention, the term "treatment" means any action in which symptoms of diseases such as cancer are improved or advantageously changed by administration of the composition according to the present invention.

The term "administration" in the present invention means to provide the composition of the present invention to a subject in any suitable way.

The term "individual" in the present invention means a subject to which the composition of the present invention can be administered in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, dog, cat , Horses, and cows.

The term "pharmaceutical composition" in the present invention means that it is manufactured for the purpose of preventing or treating cancer, and can be used by being formulated in various forms according to a conventional method. For example, powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc. can be formulated into oral dosage forms, creams, gels, patches, sprays, ointments, warnings, lotions, linen agents, pasta agents, or It can be formulated and used in the form of external preparations for skin, suppositories and sterile injectable solutions such as cataplasma.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. At this time, carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) Or by mixing lactose, gelatin, and the like. In addition, lubricants such as magnesium styrene talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, flavoring agents, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage may be adjusted according to the patient's condition, weight, and disease. It depends on the degree, drug type, route of administration, and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of discharge, duration of treatment, factors including co-drugs, and other factors well known in the medical field can be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art. The administration may be administered once a day, or may be divided into several times.

In the present invention, the pharmaceutical composition may further include an anti-cancer agent, and the anti-cancer agent may be doxorubicin, cisplatin, gemcitabine, oxaliplatin, 5-FU, etc., but is not limited to the compound used as an anti-cancer agent. Does not.

In the present invention, the pharmaceutical composition inhibits the stem cellularity of cancer stem cells, through which it is possible to suppress the proliferation, survival, metastasis, recurrence, or the development of anticancer drug resistance, but the stem cellularity of cancer stem cells If the effect can be caused by suppressing, it is not limited thereto.

In the present invention, the composition may further include a substance that blocks Wnt signal activation.

In addition, the present invention is an antibody for detecting the phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4, the 351th serine of phosphorylated mouse OCT4 Or it provides an antibody characterized in that it specifically binds to the 359th serine of phosphorylated human OCT4.

In the present invention, the term "antibody" refers to a polypeptide comprising a framework region derived from an immunoglobulin gene or fragment thereof that recognizes by specifically binding to an antigen. Recognized immunoglobulin genes include a myriad of immunoglobulin variable region genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains are classified as kappa or lambda, and heavy chains are classified as gamma, mu, alpha, delta, or epsilon, consequently defining the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody becomes key to binding specificity and affinity. In some embodiments, antibodies or fragments of antibodies can be derived from different organisms, including humans, mice, rats, hamsters, rabbits, camels, and the like.

In the present invention, the antibody for detecting the phosphorylation of the 351th serine in the amino acid sequence of mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4 may be a polyclonal antibody derived from rabbit, but is not limited thereto. The epitope recognized by (epitope) may be represented by the amino acid sequence of SEQ ID NO: 7 or 8.

In addition, the present invention provides a method for screening a stem cell inhibitory substance of stem cells, comprising the following steps:

(a) processing a candidate substance in cells expressing mouse octamer-binding transcription factor 4 (OCT4) or human OCT4;

(b) determining whether phosphorylation of the 351th serine in the amino acid sequence of the mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4; And

(c) If the serine is not phosphorylated in step (b), selecting the candidate substance as a stem cell inhibitor of stem cells.

In addition, the present invention provides a method for screening a stem cell inhibitory substance of stem cells, comprising the following steps:

(a) processing a candidate substance in cells expressing mouse octamer-binding transcription factor 4 (OCT4) or human OCT4;

(b) checking whether Wnt signal is activated in the cell; And

(c) when the Wnt signal is not activated in the step (b), selecting the candidate substance as a stem cell suppressor of stem cells.

In the present invention, the screening method comprises the steps of (b), confirming whether Wnt signal is activated in the cell; And

In the step (c), when the Wnt signal is not activated, it may include the step of selecting the candidate substance as a stem cell inhibitory substance of stem cells.

In the present invention, the candidate material may be a nucleotide, DNA, RNA, amino acid, aptamer, protein, compound, natural product, natural extract, and the like, and is not limited to any material that can be used for stem cell suppression of stem cells.

In addition, the present invention provides a method for providing information for diagnosing whether stem cells are inhibited by stem cells, comprising the following steps:

(a) determining whether phosphorylation of the 351 th serine in the amino acid sequence of mouse OCT4 or the 359 th serine in the amino acid sequence of human OCT4 from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4; And

(b) When the serine is not phosphorylated in the step (a), diagnosing that the stem cell properties of the stem cells are suppressed.

In the present invention, the information providing method comprises the steps of (a), determining whether Wnt signal is activated from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4; And

In the step (b), when the Wnt signal is not activated, it may include a step of diagnosing that stem cellity of the stem cells is suppressed.

In addition, the present invention provides a method for providing information for diagnosing whether stem cells are inhibited by stem cells, comprising the following steps:

(a) determining whether Wnt signal is activated from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4; And

(b) when Wnt signal is not activated in the step (a), diagnosing that stem cellity of the stem cells is suppressed.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example  One. OCT4  Protein phosphorylation and OCT4 activity  Check association

1-1. OCT4  Production of phosphorylated antibodies

In order to produce an antibody that can specifically recognize only OCT4 phosphorylation, an OCT4 protein sequence is provided from the National Center for Biotechnology Information (NCBI), and as shown in FIG. 1, 14 amino acid sequences containing OCT4 phosphorylation are selected to prepare the antibody. Commissioned by a professional company (GenScript Biotech Corp., New Jersey, United States).

Then, to confirm the produced antibodies, the embryonic stem cell line was washed with a phosphate buffered saline (PBS), and a cell lysis buffer solution (Lysis buffer; 20 mM Tris-Cl (pH 7.4), 150 mM NaCl , 1mM EDTA, 1% (v / v) Triton X-100 and Protease inhibitor (# 78415, Thermo Scientific)), and antibody produced after cell lysis and OCT4 antibody (# sc-5279, SantaCruz), and It was confirmed by SDS-PAGE and Western blotting using ACTB antibody (# ab8227, Abcam).

In order to confirm that only the phosphorylation of the serine 351 position of OCT4 is specifically recognized, when the protein produced during Western blotting of the protein of the embryonic stem cell is recognized with the peptide containing the phosphorylation position, it is confirmed whether it inhibits cognition. Did. As a result, as shown in FIG. 2, when the peptide containing the phosphorylation site was recognized with the antibody together, phosphorylation of the 351th serine of OCT4 was not recognized, and the peptide was not recognized together or the peptide at a position other than the phosphorylation site When it was recognized together, it was confirmed that phosphorylation was recognized.

In addition, as a result of confirming whether OCT4 and OCT4 phosphorylation were performed according to the presence or absence of doxycycline in an embryonic stem cell line that artificially inhibits the expression of resistant OCT4 by treating doxycycline, phosphorylation of OCT4 was not recognized when treated with doxycycline as shown in FIG. No, it was confirmed that it is recognized when not treated.

In addition, it was confirmed whether the antibody produced above recognizes OCT4 phosphorylation using immunofluorescence staining. To this end, the embryonic stem cell line was washed with a phosphate buffer solution, and then the cells were fixed using a 4% formaldehyde (# F1635, Sigma) solution, followed by washing with a phosphate buffer solution and 0.5% Triton X -100 (# 069, Amresco) was treated to increase the permeability of the cell membrane. Then, blocking was performed using a 1% Bovine Serum Albumin (BSA, # A0100-050, GeneDEPOT) solution, and the peptides containing the phosphorylation site and the non-phosphorylation site were treated with OCT4 phosphorylated antibody at room temperature for 2 hours. To react. Thereafter, after removing the unbound antibody with a phosphate buffer solution, the fluorescently bound secondary antibody (IgG-FITC, # 31569, Thermo Fisher Scientific) is treated and reacted at room temperature for 1 hour. In addition, it was removed using a phosphate buffer solution, and the nuclei were stained using 4 ', 6-diamidino-2-phenylindole (DAPI, #CAS 28718-90-3, Calbiochem). Then, after the mounting solution was added onto the cells, protein expression was confirmed using a confocal microscope. As a result, as shown in FIG. 4, when the peptide containing the phosphorylation site was recognized with the antibody, phosphorylation of the 351th serine of OCT4 was not recognized, and when the peptide at a position other than the phosphorylation site was recognized together, phosphorylation Was confirmed.

In addition, it was confirmed by immunoprecipitation (Immunoprecipitation) to determine whether the recognized OCT4 phosphorylation is derived from OCT4 protein. First, the OCT4 protein was precipitated using the OCT4 antibody in the cell lysate of the embryonic stem cell line, and the result was confirmed by Western blotting using the OCT4 phosphorylated antibody. As a result, it was confirmed that the phosphorylation of the recognized OCT4 is derived from the OCT4 protein, as shown in FIG. 5.

1-2. In human cancer cells OCT4  Phosphorylation confirmation

It was confirmed that the phosphorylation of the 351 th serine of OCT4 recognized in Example 1-1 was detected not only in the embryonic stem cell line but also in human cancer cells. To this end, NCCIT and NTERA2 cell lines with embryonic stem cell characteristics among human cancer cells were used, mouse embryonic stem cell (E14) as a positive control, and human fetal kidney cell line (293FT) as a negative control. As a result of the Western blotting method of Example 1-1, it was observed that phosphorylation of OCT4 was detected in human cancer cells as shown in FIG. 6.

1-3. OCT4  With phosphorylation OCT4  Check active association

The relationship between phosphorylation of OCT4 and OCT4 activity can be confirmed by a method of evaluating embryonic stem cell maintenance. To accomplish this, first, the non-phosphorylated mutant S351A, which substituted the 351th serine of OCT4 with alanine, and the S351E, a phosphorylated imitation mutant, which substituted 351th serine of OCT4 with glutamic acid, were prepared through site-directed mutatgenesis. Did. Thereafter, OCT4 inherent in the embryonic stem cells was removed, and a new cell line expressing OCT4 normal or mutants was prepared. For cell production, expressing OCT4 and puromycin resistance genes substituted with OCT4 wild type and puromycine resistance genes or non-phosphorylated / mimetic S351A / S351E of OCT4 in pMSCV-FLAG-puro vector A retrovirus was produced and transfected with the mouse embryonic stem cell line ZHBTc4, which can inhibit the expression of endogenous OCT4 when treated with doxycycline. Then, the medium was treated with puromycin to isolate the normally transfected cell line, and the isolated cell line was treated with doxycycline so that the expression of endogenous OCT4 was inhibited and the substituted exogenous OCT4 protein was expressed. To confirm that the same dose of retrovirus was transfected, the mouse was infected with NIH3T3, a mouse fibroblast line that does not express OCT4. Protein expression was confirmed by Western blotting with OCT4 antibody (# sc-5279, SantaCruz), and ACTB antibody (# ab8227, Abcam) was used as a control.

As a result, as shown in FIG. 7, it was confirmed that OCT4 substituted with non-phosphorylated / mimetic S351A / S351E in the NIH3T3 cell line was expressed in an amount similar to wild type (WT).

In addition, prior to treatment with doxycycline, the cell line was washed with a phosphate buffered saline (PBS), and a cell cluster was separated into single cells using TE (Trypsin-EDTA, # 15400054, Thermo Fisher). After that, the cell culture medium was added so that the final cell lysate had a volume of 1 ml. Thereafter, 100 ul of cell lysate, a volume of 1/10 per well, was dispensed into a 6-well plate and treated with doxycycline and puromycin, and cultured for 5 to 7 days at 37 ° C and 5% CO ₂ conditions. The cultured cells were stained using an alkaline phosphatase assay kit (Medsource Ozone Biomedicals), and observed under a microscope.

As a result, as shown in FIG. 8, in the case of the non-phosphorylated mutant cell line of OCT4 (S351A), the number of embryonic stem cells forming colonies decreases when compared to the control wild-type cell line, whereas the phosphorylated mimic cell line of OCT4 (S351E) confirmed that the number of colonies was recovered as much as the wild-type cell line.

In addition, a cell line stably expressing the OCT4 mutant protein was prepared to confirm the effect of OCT4 phosphorylation on embryonic stem cells. To accomplish this, the pCAG-Flag vector was recombined to express the OCT4 wild type and puromycin resistance genes, or the OCT4 and puromycin resistance genes substituted with S351A / S351E, which are non-phosphorylated / mimetic forms of OCT4. The plasmid was transfected into the mouse embryonic stem cell line ZHBTc4, which can inhibit the expression of endogenous OCT4 when treated with doxycycline using Lipofectamine 3000 (Thermefisher). Then, the cell line that was normally transfected was separated by treatment with puromycin in the medium, and the expression of endogenous OCT4 was inhibited by treating doxycycline in the separated cell line, and the cell line substituted with exogenous OCT4 protein (ZHBTc4 OCT4-WT, ZHBTc4 OCT4-S351A) , ZHBTc4 OCT4-S351E). The produced cell lines were confirmed by Western blotting. Cell lines treated with doxycycline and cultured for 4 to 8 days were washed with a phosphate buffered saline (PBS), lysis buffer (20 mM Tris-Cl (pH 7.4), 150 mM NaCl, Cells were lysed using 1 mM EDTA, 1% (v / v) Triton X-100 and protease inhibitors, and then confirmed by SDS-PAGE and Western blotting using OCT4 antibody and FLAG antibody (# F3165, Sigma). . ACTB antibody was used as a control.

As a result, as shown in FIG. 9, it was confirmed that OCT4 substituted with wild type OCT4 or S351A / S351E was normally transformed, and that the exogenous OCT4 protein was expressed by the amount of endogenous OCT4 protein depending on doxycycline.

In addition, the cell lines prepared above were cultured for 5 to 7 days at 37 ° C and 5% CO ₂ conditions while dispensing 1000 single cells per well into 6-well plates and treating doxycycline and puromycin. The cultured cells were stained using an alkaline phosphatase assay kit (Medsource Ozone Biomedicals), and observed under a microscope.

As a result, as shown in FIG. 10, in the case of cells stably expressing non-phosphorylated OCT4 (S351A), the cells have a broadly spread cell shape compared to the control wild type, while expressing phosphorylated mimic OCT4 (S351E). The cells were confirmed to have the same dome shape as the control cells.

In addition, stem cell self-renewal ability was evaluated using SSEA-1 antibody, which is a marker for stem cell self-renewal ability. To this end, after dispensing 1000 single cells per well into a 6-well plate and treating doxycycline and puromycin, cells cultured for 5 to 7 days at 37 ° C and 5% CO ₂ conditions were stained with SSEA-1. It was confirmed by flow cytometry (Fluorescence activated cell sorting, FACS).

As a result, as shown in FIG. 11, ZHBTc4 OCT4-S351E, a cell line expressing phosphorylated imitation OCT4 (S351E), was stained in the same way as wild type (ZHBTc4 OCT4-WT), but SSEA-1 was stained, but was not phosphorylated OCT4 (S351A) expressing cell line ZHBTc4 OCT4-S351A was less stained.

Example  2. OCT4  With phosphorylation Wnt  Check signal conditioning association

2-1. OCT4  Evaluation of the effect of phosphorylation on embryonic stem cells

The embryonic stem cell lines stably expressing the exogenous OCT4 produced by the method of Example 1-3 were treated with doxycycline in the ZHBTc4 OCT4-WT, ZHBTc4 OCT4-S351A, and the cultured cells were used with the RNeasy Purification Kit (# 74104, Qiagen). After the total RNA was purified, it was commissioned to a specialized analysis company (Macrogen, Korea) to perform total RNA-sequencing. For the entire RNA-sequencing result, genes regulated directly by OCT4 were selected using published ChIP data. The results are shown in FIG. 12.

Path analysis was performed on the genes selected from the above, and the results were visualized using a visualization program (Cytoscape, 3.6.1). As a result of the pathway analysis, it was confirmed that the pathway related to the starch differentiation ability of the stem cells and the Wnt signal pathway are changed as shown in FIG. 13. In particular, when the non-phosphorylated mutant OCT4 is expressed by Gene Set Enrichment Analysis (GSEA) for the Wnt signal pathway, it was confirmed that the Wnt signal is inhibited, as shown in FIG. 14.

2-2. OCT4  With phosphorylation Wnt  Check signal association

The cell line produced in the same manner as in Example 1-3 is dispensed 1000 single cells per well into a 6-well plate, and WNT3A, a recombinant protein capable of artificially activating doxycycline, puromycin, and Wnt signals, or the compound CHIR9901 While incubating, cultured at 37 ° C and 5% CO ₂ for 5 to 7 days. The cultured cells were stained using an alkaline phosphatase assay kit and observed under a microscope.

As a result, as shown in FIG. 15, it was confirmed that the stem cell self-renewal ability is restored by artificially activating the Wnt signal in cells stably expressing non-phosphorylated OCT4.

In addition, the stem cell self-renewal capability marker SSEA-1 antibody was used to evaluate the stem cell self-renewal capability, and as a result, it was also confirmed that self-renewal capability is restored when Wnt signal is activated as shown in FIG. 16.

Example  3. OCT4  Phosphorylation-induced kinase ( kinase ) Confirm

To confirm the association between CSNK2A1 and OCT4 phosphorylation as a kinase that induces phosphorylation of the 351th serine of OCT4, a cell line for regulating CSNK2A1 expression inhibition was prepared. To this end, a lentivirus expressing the shRNA of SEQ ID NO: 5 or SEQ ID NO: 6 dependently on doxycycline was produced and transfected into the embryonic stem cell line E14 to prepare a cell line capable of regulating the expression of CSNK2A1 through doxycycline. Then, as a result of Western blotting in the same manner as in Example 1-1, it was confirmed that the kinase that induces phosphorylation of OCT4 is CSNK2A1 as shown in FIG. 17.

In addition, an in vitro kinase assay was performed using GST fusion recombinant OCT4 protein and GST fusion recombinant CSNK2A1. Kinase reaction solution (Kinase buffer; 60mM HEPES-NaOH pH 7.5, 3mM MgCl ₂ , 3mM MnCl ₂ , 3μM Na-orthovanadate, with 1 μg of GST Fusion OCT4 recombinant protein and 0.2ug of GST Fusion CSNK2A1 recombinant protein with 0.5mM ATP) 1.2mM DTT) was mixed and reacted at 30 ° C for 30 minutes, followed by Western blotting with SDS-PAGE. As a result, as shown in FIG. 18, when OCT4 and CSNK2A1 were present together, the phosphorylated antibody was confirmed to recognize phosphorylation of OCT4.

The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.

<110> National Cancer Center <120> Composition for inhibiting stemness of stem cell comprising          material for inhibiting phosphorylation of OCT4 and blocking          activation of Wnt signal <130> MP18-214 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 352 <212> PRT <213> Mus musculus <220> <221> PEPTIDE <222> (1) .. (352) <223> POU domain, class 5, transcription factor 1 isoform 1 <400> 1 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly   1 5 10 15 Gly Gly Asp Gly Ser Ala Gly Leu Glu Pro Gly Trp Val Asp Pro Arg              20 25 30 Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly Pro          35 40 45 Gly Ser Glu Val Leu Gly Ile Ser Pro Cys Pro Pro Ala Tyr Glu Phe      50 55 60 Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val Gly Leu Gly Leu Val  65 70 75 80 Pro Gln Val Gly Val Glu Thr Leu Gln Pro Glu Gly Gln Ala Gly Ala                  85 90 95 Arg Val Glu Ser Asn Ser Glu Gly Thr Ser Ser Glu Pro Cys Ala Asp             100 105 110 Arg Pro Asn Ala Val Lys Leu Glu Lys Val Glu Pro Thr Pro Glu Glu         115 120 125 Ser Gln Asp Met Lys Ala Leu Gln Lys Glu Leu Glu Gln Phe Ala Lys     130 135 140 Leu Leu Lys Gln Lys Arg Ile Thr Leu Gly Tyr Thr Gln Ala Asp Val 145 150 155 160 Gly Leu Thr Leu Gly Val Leu Phe Gly Lys Val Phe Ser Gln Thr Thr                 165 170 175 Ile Cys Arg Phe Glu Ala Leu Gln Leu Ser Leu Lys Asn Met Cys Lys             180 185 190 Leu Arg Pro Leu Leu Glu Lys Trp Val Glu Glu Ala Asp Asn Asn Glu         195 200 205 Asn Leu Gln Glu Ile Cys Lys Ser Glu Thr Leu Val Gln Ala Arg Lys     210 215 220 Arg Lys Arg Thr Ser Ile Glu Asn Arg Val Arg Trp Ser Leu Glu Thr 225 230 235 240 Met Phe Leu Lys Cys Pro Lys Pro Ser Leu Gln Gln Ile Thr His Ile                 245 250 255 Ala Asn Gln Leu Gly Leu Glu Lys Asp Val Val Arg Val Trp Phe Cys             260 265 270 Asn Arg Arg Gln Lys Gly Lys Arg Ser Ser Ile Glu Tyr Ser Gln Arg         275 280 285 Glu Glu Tyr Glu Ala Thr Gly Thr Pro Phe Pro Gly Gly Ala Val Ser     290 295 300 Phe Pro Leu Pro Pro Gly Pro His Phe Gly Thr Pro Gly Tyr Gly Ser 305 310 315 320 Pro His Phe Thr Thr Leu Tyr Ser Val Pro Phe Pro Glu Gly Glu Ala                 325 330 335 Phe Pro Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser Asn             340 345 350 <210> 2 <211> 360 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) .. (360) <223> POU domain, class 5, transcription factor 1 isoform 1 <400> 2 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly   1 5 10 15 Gly Gly Gly Asp Gly Pro Gly Gly Pro Glu Pro Gly Trp Val Asp Pro              20 25 30 Arg Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly          35 40 45 Pro Gly Val Gly Pro Gly Ser Glu Val Trp Gly Ile Pro Pro Cys Pro      50 55 60 Pro Pro Tyr Glu Phe Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val  65 70 75 80 Gly Val Gly Leu Val Pro Gln Gly Gly Leu Glu Thr Ser Gln Pro Glu                  85 90 95 Gly Glu Ala Gly Val Gly Val Glu Ser Asn Ser Asp Gly Ala Ser Pro             100 105 110 Glu Pro Cys Thr Val Thr Pro Gly Ala Val Lys Leu Glu Lys Glu Lys         115 120 125 Leu Glu Gln Asn Pro Glu Glu Ser Gln Asp Ile Lys Ala Leu Gln Lys     130 135 140 Glu Leu Glu Gln Phe Ala Lys Leu Leu Lys Gln Lys Arg Ile Thr Leu 145 150 155 160 Gly Tyr Thr Gln Ala Asp Val Gly Leu Thr Leu Gly Val Leu Phe Gly                 165 170 175 Lys Val Phe Ser Gln Thr Thr Ile Cys Arg Phe Glu Ala Leu Gln Leu             180 185 190 Ser Phe Lys Asn Met Cys Lys Leu Arg Pro Leu Leu Gln Lys Trp Val         195 200 205 Glu Glu Ala Asp Asn Asn Glu Asn Leu Gln Glu Ile Cys Lys Ala Glu     210 215 220 Thr Leu Val Gln Ala Arg Lys Arg Lys Arg Thr Ser Ile Glu Asn Arg 225 230 235 240 Val Arg Gly Asn Leu Glu Asn Leu Phe Leu Gln Cys Pro Lys Pro Thr                 245 250 255 Leu Gln Gln Ile Ser His Ile Ala Gln Gln Leu Gly Leu Glu Lys Asp             260 265 270 Val Val Arg Val Trp Phe Cys Asn Arg Arg Gln Lys Gly Lys Arg Ser         275 280 285 Ser Ser Asp Tyr Ala Gln Arg Glu Asp Phe Glu Ala Ala Gly Ser Pro     290 295 300 Phe Ser Gly Gly Pro Val Ser Phe Pro Leu Ala Pro Gly Pro His Phe 305 310 315 320 Gly Thr Pro Gly Tyr Gly Ser Pro His Phe Thr Ala Leu Tyr Ser Ser                 325 330 335 Val Pro Phe Pro Glu Gly Glu Ala Phe Pro Pro Val Ser Val Thr Thr             340 345 350 Leu Gly Ser Pro Met His Ser Asn         355 360 <210> 3 <211> 1353 <212> RNA <213> Mus musculus <220> <221> gene <222> (1) .. (1353) <223> POU domain, class 5, transcription factor 1 (Pou5f1), transcript          variant 1, mRNA <400> 3 gaggtgaaac cgtccctagg tgagccgtct ttccaccagg cccccggctc ggggtgccca 60 ccttccccat ggctggacac ctggcttcag acttcgcctt ctcaccccca ccaggtgggg 120 gtgatgggtc agcagggctg gagccgggct gggtggatcc tcgaacctgg ctaagcttcc 180 aagggcctcc aggtgggcct ggaatcggac caggctcaga ggtattgggg atctccccat 240 gtccgcccgc atacgagttc tgcggaggga tggcatactg tggacctcag gttggactgg 300 gcctagtccc ccaagttggc gtggagactt tgcagcctga gggccaggca ggagcacgag 360 tggaaagcaa ctcagaggga acctcctctg agccctgtgc cgaccgcccc aatgccgtga 420 agttggagaa ggtggaacca actcccgagg agtcccagga catgaaagcc ctgcagaagg 480 agctagaaca gtttgccaag ctgctgaagc agaagaggat caccttgggg tacacccagg 540 ccgacgtggg gctcaccctg ggcgttctct ttggaaaggt gttcagccag accaccatct 600 gtcgcttcga ggccttgcag ctcagcctta agaacatgtg taagctgcgg cccctgctgg 660 agaagtgggt ggaggaagcc gacaacaatg agaaccttca ggagatatgc aaatcggaga 720 ccctggtgca ggcccggaag agaaagcgaa ctagcattga gaaccgtgtg aggtggagtc 780 tggagaccat gtttctgaag tgcccgaagc cctccctaca gcagatcact cacatcgcca 840 atcagcttgg gctagagaag gatgtggttc gagtatggtt ctgtaaccgg cgccagaagg 900 gcaaaagatc aagtattgag tattcccaac gagaagagta tgaggctaca gggacacctt 960 tcccaggggg ggctgtatcc tttcctctgc ccccaggtcc ccactttggc accccaggct 1020 atggaagccc ccacttcacc acactctact cagtcccttt tcctgagggc gaggcctttc 1080 cctctgttcc cgtcactgct ctgggctctc ccatgcattc aaactgaggc accagccctc 1140 cctggggatg ctgtgagcca aggcaaggga ggtagacaag agaacctgga gctttggggt 1200 taaattcttt tactgaggag ggattaaaag cacaacaggg gtggggggtg ggatggggaa 1260 agaagctcag tgatgctgtt gatcaggagc ctggcctgtc tgtcactcat cattttgttc 1320 ttaaataaag actgggacac acagtagata gct 1353 <210> 4 <211> 1430 <212> RNA <213> Homo sapiens <220> <221> gene <222> (1) .. (1430) <223> POU class 5 homeobox 1 (POU5F1), transcript variant 1, mRNA <400> 4 agagaggggt tgagtagtcc cttcgcaagc cctcatttca ccaggccccc ggcttggggc 60 gccttccttc cccatggcgg gacacctggc ttcggatttc gccttctcgc cccctccagg 120 tggtggaggt gatgggccag gggggccgga gccgggctgg gttgatcctc ggacctggct 180 aagcttccaa ggccctcctg gagggccagg aatcgggccg ggggttgggc caggctctga 240 ggtgtggggg attcccccat gccccccgcc gtatgagttc tgtgggggga tggcgtactg 300 tgggccccag gttggagtgg ggctagtgcc ccaaggcggc ttggagacct ctcagcctga 360 gggcgaagca ggagtcgggg tggagagcaa ctccgatggg gcctccccgg agccctgcac 420 cgtcacccct ggtgccgtga agctggagaa ggagaagctg gagcaaaacc cggaggagtc 480 ccaggacatc aaagctctgc agaaagaact cgagcaattt gccaagctcc tgaagcagaa 540 gaggatcacc ctgggatata cacaggccga tgtggggctc accctggggg ttctatttgg 600 gaaggtattc agccaaacga ccatctgccg ctttgaggct ctgcagctta gcttcaagaa 660 catgtgtaag ctgcggccct tgctgcagaa gtgggtggag gaagctgaca acaatgaaaa 720 tcttcaggag atatgcaaag cagaaaccct cgtgcaggcc cgaaagagaa agcgaaccag 780 tatcgagaac cgagtgagag gcaacctgga gaatttgttc ctgcagtgcc cgaaacccac 840 actgcagcag atcagccaca tcgcccagca gcttgggctc gagaaggatg tggtccgagt 900 gtggttctgt aaccggcgcc agaagggcaa gcgatcaagc agcgactatg cacaacgaga 960 ggattttgag gctgctgggt ctcctttctc agggggacca gtgtcctttc ctctggcccc 1020 agggccccat tttggtaccc caggctatgg gagccctcac ttcactgcac tgtactcctc 1080 ggtccctttc cctgaggggg aagcctttcc ccctgtctcc gtcaccactc tgggctctcc 1140 catgcattca aactgaggtg cctgcccttc taggaatggg ggacaggggg aggggaggag 1200 ctagggaaag aaaacctgga gtttgtgcca gggtttttgg gattaagttc ttcattcact 1260 aaggaaggaa ttgggaacac aaagggtggg ggcaggggag tttggggcaa ctggttggag 1320 ggaaggtgaa gttcaatgat gctcttgatt ttaatcccac atcatgtatc acttttttct 1380 taaataaaga agcctgggac acagtagata gacacactta aaaaaaaaaa 1430 <210> 5 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> shRNA # 1 of CSNK2A1 <400> 5 ccggccgagt tgcttctcga tatttctcga gaaatatcga gaagcaactc ggttttg 57 <210> 6 <211> 58 <212> RNA <213> Artificial Sequence <220> <223> shRNA # 2 of CSNK2A1 <400> 6 ccggacagac tatgacattc gatttctcga gaaatcgaat gtcatagtct gttttttg 58 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> epitope of OCT4 <400> 7 Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser Asn   1 5 10 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> epitope of OCT4 <400> 8 Pro Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser   1 5 10

Claims (24)

A composition for inhibiting stem cell stem cellity, comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4.
According to claim 1,
The composition is characterized in that it further comprises a substance that blocks Wnt signal activation, stem cell composition for stem cell suppression.
According to claim 1,
The amino acid sequence of the mouse OCT4 is characterized in that represented by the amino acid sequence of SEQ ID NO: 1, stem cell stem cell composition for inhibiting.
According to claim 1,
The amino acid sequence of the human OCT4 is characterized in that represented by the amino acid sequence of SEQ ID NO: 2, stem cell stem cell composition for inhibiting.
According to claim 1,
The material is a casein kinase 2 alpha 1 (casein kinase 2 alpha 1; CSNK2A1), characterized in that it comprises a substance that inhibits the expression, stem cell stem cell composition for inhibiting stem cells.
The method of claim 5,
The substance that inhibits the expression of CSNK2A1 is a short hairpin RNA (shRNA), represented by the nucleotide sequence of SEQ ID NO: 5 or 6, stem cell stem cell composition for inhibiting stem cells.
According to claim 1,
The stem cell is characterized in that any one selected from the group consisting of embryonic stem cells, germ cells, and cancer stem cells, stem cell stem cell composition for inhibiting.
According to claim 1,
The composition is characterized in that to inhibit the proliferation, self-renewal, colonization, or survival of stem cells, stem cell composition for inhibiting stem cells.
A kit for stem cell regulation of stem cells, comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 and a Wnt signal activator .
A cell therapeutic agent comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4 and stem cells for cell therapy as an active ingredient.
The method of claim 10,
The cell therapy, characterized in that it further comprises a substance that blocks Wnt signal activation, cell therapy.
The method of claim 10,
The cell therapy is characterized in that to inhibit the metastasis, survival, or cancer of the stem cells for treatment, cell therapy.
In vitro , characterized in that it comprises the step of inhibiting the phosphorylation of the amino acid sequence of the mouse OCT4 (octamer-binding transcription factor 4) 351 serine or human amino acid sequence 359 serine, the control of OCT4 phosphorylation function Through stem cell suppression method.
The method of claim 13,
The stem cell suppression method further comprises the step of blocking Wnt signal activation, stem cell suppression method through OCT4 phosphorylation function control.
In vitro , inhibiting phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4; And activating the Wnt signal, a method for regulating stem cells through OCT4 phosphorylation.
A pharmaceutical composition for preventing or treating cancer, comprising a substance that inhibits phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4.
The method of claim 16,
The pharmaceutical composition is characterized in that it further comprises an anti-cancer agent, pharmaceutical composition.
The method of claim 16,
The pharmaceutical composition is characterized in that to inhibit the proliferation, survival, metastasis, relapse or anticancer drug resistance of cancer, pharmaceutical composition.
The method of claim 16,
The composition is characterized in that it further comprises a substance that blocks Wnt signal activation, pharmaceutical composition.
As an antibody for detecting the phosphorylation of the 351th serine in the amino acid sequence of mouse octamer-binding transcription factor 4 (OCT4) or the 359th serine in the amino acid sequence of human OCT4,
The antibody recognizes the epitope represented by the amino acid sequence of SEQ ID NO: 7 or 8 and is characterized in that it specifically binds to the 351th serine of phosphorylated mouse OCT4 or the 359th serine of phosphorylated human OCT4. Antibodies.
A method for screening a stem cell inhibitory substance of a stem cell, comprising the following steps:
(a) processing a candidate substance in cells expressing mouse octamer-binding transcription factor 4 (OCT4) or human OCT4;
(b) determining whether phosphorylation of the 351th serine in the amino acid sequence of the mouse OCT4 or the 359th serine in the amino acid sequence of human OCT4; And
(c) If the serine is not phosphorylated in step (b), selecting the candidate substance as a stem cell inhibitor of stem cells.
The method of claim 21,
The screening method comprises the steps (b), confirming whether Wnt signal is activated in the cell; And
In the step (c), when the Wnt signal is not activated, comprising the step of selecting the candidate substance as a stem cell inhibitor of stem cells, stem cell screening method of stem cells inhibitory substances .
A method of providing information for diagnosing whether stem cells are inhibited by stem cells, including the following steps:
(a) determining whether phosphorylation of the 351 th serine in the amino acid sequence of mouse OCT4 or the 359 th serine in the amino acid sequence of human OCT4 from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4; And
(b) When the serine is not phosphorylated in the step (a), diagnosing that the stem cell properties of the stem cells are suppressed.
The method of claim 23,
The method for providing information is to determine whether Wnt signal is activated from cells expressing mouse OCT4 (octamer-binding transcription factor 4) or human OCT4 in the step (a); And
In the step (b), when the Wnt signal is not activated, characterized in that it comprises the step of diagnosing stem cell stem cell suppression, providing information for diagnosing stem cell stem cell suppression. Way.

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