WO2015016475A1 - Pharmaceutical use of c-yes for differentiation therapy of cancer stem cells - Google Patents

Abstract

The present invention relates to the pharmaceutical use of c-Yes for differentiation therapy of cancer stem cells. More specifically, c-Yes has a remarkably high expression amount in CD133-positive cancer stem cells when compared with CD133-negative cancer stem cells, promotes the differentiation of cancer stem cells upon depletion of the c-Yes, and suppresses the proliferation of cancer stem cells so as to suppress resistance to chemotherapy or radiation therapy, or recurrence of cancer, thereby preventing or treating CD133-positive cancer.

Description

Pharmaceutical Use of c-Yes for Differentiation Treatment of Cancer Stem Cells

The present invention relates to the differentiation treatment of cancer stem cells using c-Yes depletion to reduce the stem cell properties of cancer stem cells.

Colorectal cancer is one of the leading causes of death in developed countries. Recent studies have found that some of the tumor cells cause tumor initiation, recurrence and metastasis. These cells have traits similar to those of normal stem cells and are called cancer stem cells (CSCs).

Cancer stem cells are small parts of tumor cells that have greater capacity to maintain and initiate tumor formation while promoting metastasis. They are known to have normal stem cells and other properties such as carcinogenicity, drug resistance, long lifespan, pluripotency, self-replication and the like. CSCs have unique phenotypes and functional features that help induce and maintain tumors.

A single marker, CD133 (prominin 1), is a type of trans membrane glycoprotein that appears in brain cancer, colon cancer, hematopoietic stem, endothelial progenitor cells, glioblastoma, neuronal and glial stem cells, and various pediatric brain tumors or pancreatic cancer. It is used as an index of carcinogenic tumor stem cells. These glycoproteins appear to be phosphorylated by Src-family kinsases. A large number of evidence suggests that the ratio of CD133 cells is related to tumor aggressiveness, metastasis and their resistance to chemotherapy and radiotherapy as well.

Since CSCs are highly resistant to standard chemotherapy, targeting these cells to block the cause of the problem seems to be a new paradigm.

The proto-oncogene, c-Yes (v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1), is a Src-family that is an important non-receptor tyrosine kinase involved in various signaling pathways. It is a subfamily of kinases. Src-family kinases phosphorylate tyrosine residues to induce a variety of signaling mechanisms. Src family kinases, including c-Yes, are activated in colorectal cancer and affect tumor development and progression. It is known to control self-replicating in normal stem cells. The frequent activation of c-Yes along with viral homologues in human cancer suggests that they are involved in initiating or progressing malignant phenotypes. In addition, c-Yes is upregulated in cancer cells compared to normal cells.

An object of the present invention is to provide a pharmaceutical use for the treatment of differentiation of cancer stem cells by elucidating the role of c-Yes in the regulation of proliferation or differentiation of cancer stem cells.

In order to achieve the above object, the present invention provides a composition for controlling the division or differentiation of cancer stem cells, including c-Yes or its inhibitor as a division or differentiation regulator.

The present invention also provides a method for detecting a CD133 positive cancer diagnostic marker having cancer stem cells by measuring the expression level of c-Yes in a human cancer cell sample in order to provide information necessary for diagnosing CD133 positive cancer cells having cancer stem cells. To provide.

The present invention also provides a composition for the prevention or treatment of CD133 positive cancer comprising a c-Yes inhibitor.

The present invention also provides a method for screening a medicament for preventing or treating a CD133 positive cancer comprising contacting a c-Yes gene with a candidate outside of the human body and determining whether the candidate promotes or inhibits expression of the gene. do.

The present invention also provides a method for screening a medicament for preventing or treating a CD133 positive cancer, comprising contacting a c-Yes protein with a candidate outside the body, and determining whether the candidate enhances or inhibits the function or activity of the protein. To provide.

According to the present invention, c-Yes has a significantly higher expression level in CD133 positive cancer stem cells than CD133 negative cancer stem cells, promotes the differentiation of cancer stem cells into cancer cells when c-Yes depletes, and promotes the proliferation of cancer stem cells. By inhibiting the loss of stem cell characteristics, it is possible to prevent or treat CD133 positive cancers by suppressing resistance or recurrence to chemotherapy or radiation therapy.

1 and 2 illustrate a treatment strategy of cancer cells including cancer stem cells.

Figure 3 is a photograph showing the morphological changes that appear during cell culture of CD133 ⁺ colorectal cancer cells or CD133 ^- colorectal cancer cells transfected with either shRNA (scrambled shRNA) or c-Yes shRNA of random sequence.

Figure 4 is a result of measuring the number of tumor spheres 400㎛ or more formed from CD133 ⁺ colorectal cancer cells or CD133 ^- colorectal cancer cells transfected with either shRNA or c-Yes shRNA of a random sequence.

FIG. 5 shows the results of measuring mRNA expression levels of cancer stem cells and cancer cell markers in CD133 ⁺ colorectal cancer cells or CD133 ⁻ colorectal cancer cells transfected with either shRNA or c-Yes shRNA of random sequence.

Figure 6 shows the results of the hourly cell viability experiments of CD133 ⁺ colorectal cancer cells or CD133 ^- colorectal cancer cells transfected with either shRNA or c-Yes shRNA of a random sequence.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

In general, cancer cells include cancer stem cells, and cancer stem cells are highly resistant to standard chemotherapy such as chemotherapy (chemotherapy) or radiation therapy (radiotherapy), so that cancer stem cells survive and proliferate even after the death of cancer cells. Causes cancer recurrence through (see FIGS. 1 and 2). Therefore, the present inventors have identified a drug target protein for the differentiation treatment of cancer stem cells in cancer stem cells that cause recurrence in anti-cancer treatment, and targets known to inhibit tumor differentiation by inhibiting the drug target protein. We tried to investigate. To this end, the present invention was completed by identifying the difference in the expression level of c-Yes in cancer cells and cancer stem cells, and confirming the proliferation and differentiation induced by inhibiting the expression of c-Yes.

Accordingly, the present invention provides a composition for regulating division or differentiation of cancer stem cells, including c-Yes or an inhibitor thereof, as a division or differentiation regulator.

According to one embodiment of the present invention, CD133 ⁺ cancer cells including a positive stem cell single marker, CD133, form tumor spheres of 400 μm or more by proliferation of cancer stem cells even in differentiation conditions. However, it can be seen that in the CD133 positive cancer cells depleted of c-Yes, tumor cells are not formed and the proliferation of cancer stem cells is inhibited (see FIGS. 3 and 4). In addition, the mRNA expression levels of markers representing stem cell and differentiated cancer cells were measured, and CD133, Nanog, and Oct4, which are characteristic markers of stem cells, were upregulated in CD133 ⁺ cells, CK-20 is significantly upregulated in CD133 ⁻ cells. In addition, depletion of c-Yes induces the expression of CK-20, a characteristic marker of cancer cells in CD133 ⁺ cells (see FIG. 5). In addition, depletion of c-Yes prolongs the midbody and increases proliferation doubling time (see FIG. 6). This is thought to cause aneuploidy by misregulation of microtubules during chromosome division. This indicates that c-Yes plays an important role in initiating and maintaining the tumorigenic properties of tumors.

In summary, inhibition (or depletion) of the c-Yes gene or protein may inhibit division of cancer stem cells and induce differentiation. On the contrary, increased gene expression of c-Yes or upregulation of proteins can promote the division of cancer stem cells and inhibit the differentiation, thereby indicating that they are factors for division or differentiation of cancer stem cells.

Therefore, c-Yes or inhibitors thereof may be used as an agent for controlling the division or differentiation of cancer stem cells.

The composition for controlling the division or differentiation of cancer stem cells of the present invention may include natural or recombinant c-Yes, c-Yes protein having a physiological activity substantially equivalent thereto, or c-Yes inhibitor. Proteins having substantially equivalent physiological activity include naturally occurring / recombinant c-Yes and functional equivalents and functional derivatives thereof.

The term "functional equivalent" refers to an amino acid sequence variant in which some or all of the amino acids of the native protein are substituted, or a portion of the amino acid is deleted or added, and has substantially the same physiological activity as the native C-YES.

By "functional derivative" is meant a protein that has been modified to increase or decrease the physicochemical properties of the c-Yes protein and has substantially the same physiological activity as the native c-Yes.

The c-Yes protein of the present invention is a protein derived from mammals, preferably humans, mice, rats, gold coins, chickens, zebradanis, frogs, bark, etc., for example GenBank accession no. It refers to a protein having a known sequence such as NP_005424.1, XP_001148240.1, XP_001087926.1, NP_001003239.1, NP_001094530.1, NP_033561.1, NP_150640.1, NP_990632.1, NP_001013288.2.

According to one embodiment of the invention, c-Yes used in the present invention is GenBank accession no. NP_005424.1 or the like can be prepared by genetic engineering methods known to those skilled in the art.

Natural protein c-Yes may be similar to that of the natural type in terms of protein activity or solubility in mammalian cells than in E. coli or insect cells when the protein is produced by genetic recombination. It is considered to be.

The recombinant c-Yes protein can be separated using a conventional column chromatography method. In addition, the degree of purification of the protein can be confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE).

The c-Yes inhibitor may include an antisense-oligonucleotide, siRNA, shRNA, miRNA, or a vector comprising the sequence complementary to the c-Yes gene; Alternatively, the antibody may be any one of antibodies specific for c-Yes protein.

The composition for regulating division or differentiation of cancer stem cells of the present invention may be added as a factor for division or differentiation during culture of cancer stem cells in vitro . For example, natural or recombinant c-Yes or inhibitors thereof may be added to divide or induce differentiation of cancer stem cells to control the increase or decrease of stem cell division or to control the number of cancer cells.

The composition for controlling division or differentiation of cancer stem cells of the present invention may further include a known differentiation inducing factor inducing differentiation of cancer stem cells in addition to the c-Yes. For example, ciliary neurotrophic factor (CNTF), bone morphogenetic proteins (BMPs), transforming growth factor (TGFα), or neuregulin-1 (Nrg1) / glial growth factor-2 (GGF2) may be used.

The present invention also provides a method for detecting a CD133 positive cancer diagnostic marker having cancer stem cells by measuring the expression level of c-Yes in a human cancer cell sample in order to provide information necessary for diagnosing CD133 positive cancer cells having cancer stem cells. It is about.

The term "CD133 positive cancer with cancer stem cells" refers to a carcinoma that expresses CD133, a positive marker of cancer stem cells. In other words, cancer stem cells contain cancer stem cells, and thus refers to a cancer species that may be resistant and relapse during chemotherapy or radiation therapy. Examples include brain cancer, colon cancer, or pancreatic cancer.

In addition, the term "CD133 positive cancer diagnostic marker having cancer stem cells" refers to a substance capable of diagnosing CD133 ⁺ cancer stem cells from CD133 ^- cancer stem cells or cancer cells, and is a positive marker of cancer stem cells. Cancer expressing CD133 expresses c-Yes having high expression levels in CD133 ⁺ cancer stem cells compared to cancer cells or CD133 ^- cancer stem cells, indicating the mRNA expression level of c-Yes or the amount of c-Yes protein. Can be used as

The expression level can be measured using oligonucleotides having sequences complementary to c-Yes, such as primers or nucleic acid probes that specifically bind to c-Yes mRNA, or antibodies specific for c-Yes protein.

By primer is meant a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable temperatures and suitable buffers (ie four different nucleoside triphosphates and polymerases). . Suitable lengths of primers can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can hybridize with the template to perform the primer-specific action. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the gene that is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. In addition, the primer according to the present invention may be used for gene amplification reactions. The amplification reaction refers to amplification of nucleic acid molecules, and the amplification reactions of such genes are well known in the art, such as, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), and Liga. Agar chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA) and the like.

The nucleic acid probe refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, which is either naturally present or artificially synthesized. Say what has been done. The probe according to the invention may be single chain, preferably oligodioxyribonucleotides. Probes of the invention may include natural dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probes of the present invention may also comprise ribonucleotides. For example, probes of the present invention may be selected from the group consisting of backbone modified nucleotides, such as peptide nucleic acids (PNA) (M. Egholm et al., Nature , 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O- alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohex Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Tityl-, propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro- , Iodo-, me -, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine.

Antibodies specific for c-Yes may be polyclonal antibodies, monoclonal antibodies, human antibodies and humanized antibodies.

Examples of such antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al., Protein Eng . 8 (10): 1057-1062 (1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments and the like.

Digestion of the antibody with papain results in two identical antigen binding fragments, each "Fab" fragment with a single antigen binding site, and the remainder "Fc" fragment. Treatment of pepsin results in an F (ab ') 2 fragment that has two antigen binding sites and still can crosslink antigen. Fv is a minimal antibody fragment comprising a complete antigen recognition and binding site. This site consists of a dimer of one heavy chain and one light chain variable region and is tightly coupled non-covalently.

Methods of preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Typically, the immunizing agent and / or adjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. The immunizing agent may be a protein of the invention or a fusion protein thereof. Injecting an immunizing agent with a protein known to be immunogenic in the mammal being immunized can be effective.

Monoclonal antibodies according to the invention can be prepared by the hybridoma method described in Kohler et al., Nature , 256: 495 (1975), or by recombinant DNA methods (e.g., US Pat. No. 4,816,576). Reference). Monoclonal antibodies are also described, for example, in Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Mol. Biol ., 222: 581-597 (1991). It can be isolated from phage antibody libraries using the techniques described.

Monoclonal antibodies in the present invention are specifically those in which a portion of the heavy and / or light chain is identical to the corresponding sequence of an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, provided that it exerts the desired activity or Although homologous, the remainder of the chain (s) includes "chimeric" antibodies that are identical or homologous to antibodies from other species or to antibodies belonging to different antibody classes or subclasses or fragments of such antibodies (Morrison et. al., Proc. Natl. Acad. Sci . USA, 81: 6851-6855 (1984)).

“Humanized” forms of non-human (eg murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof, including minimal sequences derived from non-human immunoglobulins (eg, Fv, Fab, Fab ', F (ab') 2 or other antigen binding sequence of the antibody. In most cases humanized antibodies are human immunoglobulins in which residues in the complementarity determining regions (CDRs) of the recipient have been replaced with CDR residues of species other than the human (donor antibody) such as mouse, rat or rabbit with the desired specificity, affinity and ability. (Receptor antibody). In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or in the CDR or framework sequences to be introduced. In general, humanized antibodies comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all CDR regions correspond to regions of non-human immunoglobulins, and all or substantially all FR regions Corresponds to the region of human immunoglobulin sequence. Humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr. Op. Struct. Biol . 2: 593-596 (1992)).

The method for measuring the expression level of c-Yes may be performed including a known process for separating mRNA or protein from a biological sample using known techniques.

The biological sample refers to a sample obtained from a living body in which the expression level of the gene or the protein level is different from the control group according to the incidence or progression of CD133 positive cancer, and for example, the biological sample is not limited thereto. , Tissues, cells, blood, serum, plasma, saliva and urine and the like.

Determination of the expression level of the gene is preferably to measure the level of mRNA, the method for measuring the level of mRNA reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern Blots and DNA chips, etc., but is not limited thereto.

The protein level can be measured using an antibody, in which case the c-Yes protein in the biological sample and the antibody specific thereto form a conjugate, i.e., an antigen-antibody complex, and the amount of antigen-antibody complex formed is detected. Quantitative measurements can be made through the magnitude of the signal on the label. The detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement fixation assays, FACS, protein chips, and the like.

Therefore, the present invention can determine the amount of mRNA expression or protein of the control group and the amount of mRNA expression or protein of the CD133 positive cancer suspected patient through the above-described detection method, and by comparing the degree of the expression amount with the control group Diagnostic markers of CD133 positive cancer can be detected. In other words, if the expression level of c-Yes is increased it can be seen that the cancer is CD133 positive cancer stem cells.

The present invention also relates to a composition for the prophylaxis or treatment of CD133 positive cancer comprising a c-Yes inhibitor.

According to the present invention, c-Yes is upregulated in CD133 positive cancer stem cells, and c-Yes is depleted in cancer stem cells when c-Yes inhibitors are used to inhibit the proliferation of cancer stem cells and differentiate into cancer cells. Is promoted. Therefore, the composition for preventing or treating CD133 positive cancer of the present invention can be used for differentiation therapy of cancer stem cells. In other words, since cancer stem cells lose certain cancer stem cell characteristics such as drug resistance and cancer recurrence when treated with c-Yes inhibitor, the drug resistance and recurrence of cancer are suppressed when used in combination with anticancer drug treatment or radiation therapy. Can increase.

The c-Yes inhibitor may include an agent that reduces the mRNA expression of the c-Yes gene or the expression of a protein thereof, or decreases the function or activity.

The c-Yes protein inhibitor may be a peptide or compound that binds to c-Yes protein and depletes c-Yes in cancer stem cells. Such inhibitors may be selected through screening methods exemplified below, such as protein structure analysis, and may be designed using methods known in the art.

In addition, the protein inhibitor may be used polyclonal antibodies, monoclonal antibodies, human antibodies and humanized antibodies to c-Yes protein, the definition of the antibody is as described above.

By using the antibody, depletion of c-Yes in cancer stem cells promotes differentiation into cancer cells, and inhibits proliferation of cancer stem cells, thereby preventing drug resistance and recurrence when used in combination with anticancer drug treatment or radiation therapy. CD133 positive cancer can be prevented or treated.

The function or activity inhibitor of the c-Yes protein of the present invention may be delivered using liposomes, viruses, gene guns, polymers, ultrasound, or electric shock, but is not particularly limited thereto.

The c-Yes gene may be DNA encoding them or mRNA transcribed therefrom. Thus, the inhibitor for the gene may be an inhibitor that binds to the gene itself to interfere with transcription or binds to mRNA transcribed from the gene and interferes with the translation of the mRNA.

Therefore, the inhibitor of the c-Yes gene includes all inhibitors that inhibit the expression of the c-Yes gene. For example, such inhibitor may be a peptide, nucleic acid or compound that binds to the gene. Such inhibitors may be selected through screening methods exemplified below, such as cell based screening, and may be designed using methods known in the art.

In one embodiment, the inhibitor can be an antisense-oligonucleotide, siRNA, shRNA, miRNA or a vector comprising the complementary sequence for the c-Yes gene. Such antisense-oligonucleotides, siRNAs, shRNAs, miRNAs or vectors comprising them can be produced using methods known in the art.

As used herein, "siRNA" refers to a double-chain RNA that induces RNA interference through cleavage of mRNA of a target gene, and an RNA strand of a sense sequence having the same sequence as the mRNA of the target gene and an antisense having a complementary sequence thereto. It consists of the RNA strand of the sequence.

The siRNA may include a form expressed by inserting the siRNA itself or a base sequence encoding the siRNA synthesized in vitro into the expression vector.

In the present invention, the "vector" refers to a gene construct comprising an external DNA inserted into the genome encoding the polypeptide.

A vector related to the present invention is a vector in which the nucleic acid sequence that inhibits the gene is inserted into the genome, and examples of the vector include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.

In addition, the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the c-Yes gene or fragment thereof, and can bind to the mRNA to inhibit the expression of the c-Yes gene or fragment.

In addition, the shRNA (short hairpin RNA) can be used to target the shRNA common sequence region on a human or mouse prepared according to a conventional method.

In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the pharmaceutical arts, and in particular, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, carbohydrogen phosphate, sodium chloride and zinc salts), gelatinous Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like.

In addition, the composition of the present invention may further include a lubricant, a humectant, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the composition according to the invention may be prepared in an aqueous solution for parenteral administration, preferably a buffered solution such as Hanks' solution, Ringer's solution or physically buffered saline. Can be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

The compositions of the present invention may be administered systemically or topically and may be formulated in suitable formulations by known techniques for such administration. For example, in oral administration, it can be administered by mixing with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.

Various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques. In addition, an effective amount of a c-Yes inhibitor may be administered in a form suitable for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like as a solution immediately before administration to saline or buffer.

An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to achieve the effect of preventing, suppressing or alleviating the disease.

Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. For example, in adults, when administered once or several times a day, the inhibitor of the present invention is administered once or several times a day, when the compound is 0.1ng / kg to 10g / kg, a polypeptide, In the case of protein or antibody, 0.1ng / kg ~ 10g / kg, antisense-oligonucleotide, siRNA, shRNA, miRNA can be administered at a dose of 0.01ng / kg ~ 10g / kg.

The present invention also relates to a method for screening a medicament for preventing or treating a CD133 positive cancer comprising contacting a c-Yes gene with a candidate outside the human body, and determining whether the candidate promotes or inhibits expression of the gene. will be.

The present invention also provides a method for screening a medicament for preventing or treating a CD133 positive cancer comprising contacting a c-Yes protein with a candidate outside of the human body and determining whether the candidate enhances or inhibits the function or activity of the protein. To provide.

According to the screening method of the present invention, first, a candidate substance to be analyzed may be contacted with a CD133 positive cancer cell or a cancer stem cell containing the gene or protein.

The candidate substance has the potential as a medicament that promotes or inhibits the function or activity of c-Yes protein or a substance that promotes or inhibits transcription or translation of mRNA, protein from c-Yes gene sequence according to a conventional selection method. Individual nucleic acids, proteins, peptides, other extracts or natural products, compounds, or allegedly or randomly selected.

Thereafter, the expression level of the gene, the amount of the protein or the activity of the protein can be measured in the cells treated with the candidate, and as a result of the increase, the expression amount, the amount of the protein or the activity of the protein is increased or decreased When measured, the candidate material may be determined to be a material capable of treating or preventing CD133 positive cancer.

The method of measuring the expression amount of the gene, the amount of the protein or the activity of the protein in the above may be carried out through a variety of methods known in the art, for example, but not limited to reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion) And immunoprecipitation assays.

Candidates exhibiting activity that inhibits c-Yes gene expression or inhibits protein function obtained through the screening method of the present invention may be candidates for CD133 positive cancer therapeutics. In addition, the candidate may deplete c-Yes in cancer stem cells to inhibit the proliferation of cancer stem cells and induce differentiation into cancer cells.

Such candidate CD133-positive cancer drug candidates will act as leading compounds in the development of CD133-positive cancer drugs in the future, and they will promote or inhibit the function of the c-Yes gene or proteins expressed therefrom. By modifying and optimizing its structure, it is possible to develop new CD133 positive cancer therapeutics.

Matters related to genetic engineering in the present invention are described by Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Sample Preparation

(Cell culture)

Human colorectal cancer cell line HT-29 was purchased from Korean cell bank (Seoul Korea) 2.05 mM glucose, 25 mM HEPES, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were cultured in RPMI-1640 containing (GIBCO, CA, USA) Cells were incubated at 5% CO ₂ concentration at a temperature of 37 ° C. 0.05% when cells reached 80% confluency Cells were detached using trypsin and 0.53 mM EDTA and seeded in fresh flasks.

Fluorescence activated cell sorting (FACS)

HT-29 cells were detached with TE (0.05% trypsin and 0.53 mM EDTA) and prepared for BD FACS according to the manufacturer's instructions. Cells were labeled with primary CD133 / 1PE antibody (Miltenyi Biotic) and sorted using unlabeled cells as a control. Data was analyzed according to the BD FACS Asia software provided on the system.

(c-Yes transfection / screening)

FACS sorted CD133 ⁺ and CD133 ^- HT-29 cells were transfected with c-Yes shRNA plasmid using shRNA plasmid transfection reagent (Santacruz). Cells were plated with antibiotic-free medium in 6-well plates. Cells were incubated to 70% confluency and transfected at a concentration of 1 ng / μl. Transfection was performed according to the manufacturer's instructions. 48 hours after incubation with shRNA, 5 days were selected using puromycin (1 ng / μl).

(RT-PCR)

Total RNA was isolated using 1 mL of TRIzol TRIzol (Invitrogen, Rockville, MD, USA) according to the manufacturer's instructions. cDNA was synthesized using the Superscript III First Strand Synthesis system (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Primers used for PCR were purchased from Cosmogenetech (Seoul, Korea). Target regions were amplified and electrophoresed on 2% agarose gels to separate PCR products and detected in the EtBr system using BioRad Molecular Imager ^® GelDoc ™ XR.

(Cell Viability Assay)

Transfected cells were seeded in 96-well 3 × 10 ³ cells per well and attached for 24 hours. CCK-8 (10 μl) was added to each well, incubated for 1 hour at 37 ° C., and then absorbed at 450 nm using a microplate reader to analyze cell proliferation and cytotoxicity.

(Tumor sphere formation analysis)

Sorted c-Yes depleted cells were seeded in 6-well plates at a density of 1 × 10 ³ cells per mL in serum-free RPMI designed to promote sphere formation. The medium was supplemented with 10 ng / mL fibroblast growth factor, 10 ng / mL epithelial growth factor and 2.75 ng / mL selenium (insulin-transferrin-selenium solution). Cells were supplemented with fresh medium every week. On the 21st day, the sphere of 60 micrometers or more was measured.

Figure 3 shows the results of measuring the morphological changes of the cells after transfecting colorectal cancer cells classified as either CD133 ⁺ or CD133 ^- with either shRNA (scrambled shRNA) or c-Yes shRNA of a random sequence 4 is a result of measuring and quantifying tumor spheres of 400 μm or more, and significant tumor spheres were formed in CD133 ⁺ colorectal cancer cells at 3 weeks. In addition, tumor cells were not formed in colorectal cancer cells that were CD133 positive but depleted of c-Yes. This suggests that the number of cells such as stem cells is significantly reduced in number, and that depletion of c-Yes induces differentiation of cancer stem cells.

Next, RNA and protein of colorectal cancer cells transfected with either shRNA or c-Yes shRNA of the random sequence are isolated, and then the mRNA expression levels of markers representing stem cell and differentiated HT-29 cells are determined. Measured.

As shown in FIG. 5, CD133 ^- is significantly up-regulated in colon cancer cells ^-, CD133 ^+, while in the large intestine CD133, Nanog and Oct4 cancer is up-regulated, CD133 CK-20 is compared to the colon cancer cells. From this, it can be seen that CD133, Nanog and Oct4 are specific markers representing stem cells, while CK-20 is a characteristic marker of differentiated colorectal cancer cells. In addition, the expression levels of the stem cell markers CD133, Nanog and Oct4 were significantly decreased in c-Yes depleted CD133 ⁺ colorectal cancer cells, and CK-20, a marker of differentiated colorectal cancer cells, was significantly increased. Could. From the above results, c-Yes depletion was found to induce differentiation of cancer stem cells into cancer cells.

FIG. 6 shows cell viability measurements performed on colorectal cancer cells transfected with c-Yes or random sequences of shRNAs. The OD values were measured at 24 and 48 hours.

As a result, after 24 hours, the OD value was lower in c-Yes depleted colorectal cancer cells than the control group. It is believed that incomplete division by depletion of c-Yes influenced the total cell number and rate of division. This difference was more pronounced after 48 hours. The double time of HT-29 cells is known to be about 22 hours, but in the cells depleted c-Yes, the midbody is extended, and the double time of the cells is doubled (about 48 hours). This is thought to cause aneuploidy by misregulation of microtubules during chromosome division. This indicates that c-Yes plays an important role in initiating and maintaining the tumorigenic properties of tumors.

The present invention can be used in the field of diagnosis, prevention or treatment of CD133 positive cancer.

Claims (15)

1. Composition for controlling the division or differentiation of cancer stem cells comprising c-Yes or an inhibitor thereof, as a division or differentiation regulator.
2. The method of claim 1,

   c-Yes inhibitors include antisense-oligonucleotides, siRNAs, shRNAs, miRNAs or vectors comprising the sequences complementary to the c-Yes gene; Or, a composition for controlling the division or differentiation of cancer stem cells which is any one of antibodies specific for c-Yes protein.
3. A method for detecting a CD133 positive cancer diagnostic marker having cancer stem cells by measuring the expression level of c-Yes in a human cancer cell sample to provide information necessary for diagnosing CD133 positive cancer having cancer stem cells.
4. The method of claim 3,

   CD133 positive cancers include brain cancer, colon cancer, pediatric brain tumors or pancreatic cancer.
5. A composition for preventing or treating CD133 positive cancer, comprising a c-Yes inhibitor.
6. The method of claim 5,

   CD133 positive cancer is a composition for the prevention or treatment of CD133 positive cancer, including brain cancer, colon cancer, childhood brain tumors or pancreatic cancer.
7. The method of claim 5,

   c-Yes inhibitors include antisense-oligonucleotides, siRNAs, shRNAs, miRNAs or vectors comprising the sequences complementary to the c-Yes gene; Or a composition for preventing or treating CD133 positive cancer, which is one of antibodies specific for c-Yes protein.
8. The method of claim 5,

   The composition for the prevention or treatment of CD133 positive cancer is used for the prevention or treatment of cancer stem cells (differentiation therapy) composition for the prevention or treatment of CD133 positive cancer.
9. The method of claim 5,

   The composition for the prevention or treatment of CD133 positive cancer is a composition for the prevention or treatment of CD133 positive cancer that is used as a combined anticancer drug treatment or radiation therapy.
10. A method for screening a medicament for preventing or treating a CD133 positive cancer, comprising contacting a c-Yes gene with a candidate outside of the human body and determining whether the candidate promotes or inhibits expression of the gene.
11. The method of claim 10,

    Reducing the expression of the c-Yes gene is determined to be a prophylactic or therapeutic agent for cancer, wherein the therapeutic agent inhibits proliferation of cancer stem cells and induces differentiation.
12. The method of claim 11,

    CD133 positive cancer is a method for screening a medicament for preventing or treating CD133 positive cancer, including brain cancer, colorectal cancer, childhood brain tumor or pancreatic cancer.
13. A method for screening a medicament for preventing or treating a CD133 positive cancer comprising contacting a c-Yes protein with a candidate outside of the human body and determining whether the candidate enhances or inhibits the function or activity of the protein.
14. The method of claim 13,

    Inhibiting the function or activity of the c-Yes protein is determined as a prophylactic or therapeutic agent for cancer, wherein the therapeutic agent inhibits proliferation of cancer stem cells and induces differentiation. .
15. The method of claim 13,

    CD133 positive cancer is a method for screening a medicament for preventing or treating cancer, including brain cancer, colon cancer, childhood brain tumors or pancreatic cancer.

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