KR101376599B1 - Pharmaceutical composition for the treatment of cancers or inhibition of metastasis containing the expression or activity inhibitors of MAP7D2, a novel cancer therapeutic target - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, which comprises a novel cancer treatment target MAP7D2 protein expression or activity inhibitor as an active ingredient. Through the mining technique, a novel targeting anticancer candidate target was identified, and based on the gene expression pattern in the cancer cell line, an appropriate cancer cell line was selected and experimentally verified through the inhibition of the target candidate gene, through which MAP7D2 was renal cancer. Novel anticancer agents by overexpressing in various cancer tissues or cancer cells such as lung cancer, colorectal cancer, ovarian cancer, gastric cancer and liver cancer, inhibiting MAP7D2 growth, migration, invasion and metastasis of cancer cells and inducing apoptosis Since the possibility of MAP7D2 was identified as a target of MAP7D2, the expression level of MAP7D2 was used. Diagnose, it can be effectively used as an active ingredient of the therapeutic or cancer metastasis inhibiting pharmaceutical composition of the expression or activity inhibitors of protein MAP7D2 cancer.

Description

Pharmaceutical composition for the treatment of cancers or inhibition of metastasis containing the expression or activity inhibitors of MAP7D2, a novel cancer therapeutic target}

The present invention relates to a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, comprising an inhibitor for expression or activity of MAP7D2 protein, which is a novel cancer growth or cancer metastasis therapeutic target.

Cancer progression is a complex process that requires the activation of cancer oncogenes and the inactivation of tumor suppressor genes. Recently, studies using mouse models to study or treat human cancer require constant expression of certain cancer causing genes (eg, H-ras, K-ras, Myc, etc.) for tumor maintenance. It is confirmed that cancer is effectively treated by inactivation of a single cancer causing gene. This is defined as oncogene addiction, and an important goal of current cancer research is to find new methods such as fusion genomics and systems biology to find these addicted cancer-causing genes in cancer. To find an effective targeting target.

As successful targeted anticancer drugs such as Herceptin, Rituxan, Gefitinib, and Gleevec are used in clinical trials, the targeted anticancer market is growing rapidly, and multinational pharmaceutical companies are launching new targeted anticancer drugs. A lot of research and development is underway to develop. There are a number of therapeutic agents for targeted anticancer targets that are currently in preclinical or clinical trials, but efforts are still underway worldwide to acquire new targets for various cancers. A typical study is the Cancer Genome Atlas (TCGA), which is jointly conducted by NCI and NHGRI in the United States. Exon sequencing of various cancer patients finds new targets by finding important mutations frequently occurring in cancer. I'm trying to secure it.

Cancer is a heterogeneous disease whose cause and progression are very heterogeneous in morphological and pathological terms, but progress in significantly different forms at the molecular level. Recent clinical experience supports this: ERBB2 and EGFR, which are targets for successful clinical use of drugs such as Herceptin and Zephytinib, are mutated or overexpressed in only about 20-30% of all cancer patients. .

As such, since cancer progression is heterogeneous, it is necessary to acquire and analyze as many clinical samples as possible to find important targets. Currently, only a few research groups around the world have a large gene expression database and are using it to develop targeted cancer treatment targets, and many domestic studies have yet to secure large databases.

MAP7D2 (MAP7 domain containing 2) is a protein under the different names FLJ14503, MGC104944, RP11-393H10.2, and is a type of microtubule-associated protein 7 domain.

To date, studies on MAP7D2 have been directed to genes with reduced expression on the X-chromosome (US Patent Publication 2010/0029009 A1), or X-chromosome association in studies for the diagnosis of Autism spectrum disorder (ASD). Expression analysis of X-chromosome for detection of gene copy number variation in patients with chromosome X-linked mental retardation described as genes that affect the replication of about 1 Mb detected in Xp22.12 of Case 4 patients (BMCGenomics, 2007, 8: 443, 2007), specifically the information about the MAP7D2 gene, its function and its association with cancer are not known at all.

Therefore, the present inventors have conducted research to discover a target cancer treatment target, and by establishing a large-scale gene expression database of cancer tissues and through original data mining techniques, as a new target anticancer candidate target, kidney cancer, lung cancer, and colon cancer The present invention was completed by confirming that MAP7D2 overexpressed in ovarian cancer, gastric cancer, and liver cancer was significantly inhibited in the growth, infiltration and metastasis of cancer cells by inhibition of MAP7D2 expression.

Disclosure of Invention It is an object of the present invention to provide a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, comprising as an active ingredient an inhibitor of expression or activity of MAP7D2 protein, which is a novel cancer growth or cancer metastasis therapeutic target.

Still another object of the present invention is to provide a method for screening cancer growth, infiltration or metastasis inhibitors using MAP7D2.

In addition, another object of the present invention to provide a method for monitoring or diagnosing cancer in a subject using MAP7D2.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating cancer or inhibiting metastasis of cancer, comprising as an active ingredient an inhibitor of MAP7D2 (MAP7 domain containing 2) protein expression or activity.

The present invention also provides a method for screening a candidate substance for treating cancer or inhibiting cancer metastasis, comprising the following steps:

1) treating the test substance to the cell line expressing MAP7D2 protein;

2) measuring the expression level of MAP7D2 protein in the cell line; And

3) selecting the test material of which the expression level of the MAP7D2 protein is reduced compared to the control group not treated with the test material.

The present invention also provides a method for screening a candidate substance for treating cancer or inhibiting cancer metastasis, comprising the following steps:

1) treating the test substance to the MAP7D2 protein;

2) measuring the activity of the MAP7D2 protein; And

3) selecting the test substance whose activity level of the MAP7D2 protein is reduced compared to the control group not treated with the test substance.

In addition, the present invention provides a method for monitoring or diagnosing cancer using MAP7D2 protein:

1) measuring the expression level of MAP7D2 protein in a subject-derived sample; And

2) Determining that the subject with increased expression level of MAP7D2 protein than the control group as a subject at risk of cancer.

In the present invention, a large-scale gene expression database of human cancer tissue was established to discover a novel targeting anticancer candidate target MAP7D2 through data mining techniques, and the MAP7D2 is a tissue of kidney cancer, lung cancer, colorectal cancer, ovarian cancer, stomach cancer and liver cancer. Or is significantly overexpressed in cells, and inhibiting MAP7D2 significantly inhibits the growth, migration, invasion and metastasis of cancer cells and induces apoptosis, thereby inhibiting the expression or activity of MAP7D2 protein as an active ingredient. Appropriate composition can be usefully used as an active ingredient of a pharmaceutical composition for the treatment of cancer or inhibiting cancer metastasis.

1 is a diagram showing the results of analysis of the expression pattern in the clinical tissue of the MAP7D2 gene by the microarray platform U133plus2.
Figure 2 is a diagram showing the results of analyzing the expression of the MAP7D2 gene in the cancer cell line microarray platform U133plus2.
Figure 3 shows the expression pattern of colorectal cancer cell line of MAP7D2 using GAPDH as an internal control for the standardization of RNA amount.
Figure 4 is a diagram showing the expression pattern of MAP7D2 liver cancer cell line, using GAPDH as an internal control for the standardization of RNA amount.
5 is a diagram showing the expression pattern of lung cancer cell line of MAP7D2 using GAPDH as an internal control for the standardization of RNA amount.
FIG. 6 shows the results of RT-PCR to compare the expression patterns of MAP7D2 in normal and lung cancer tissues of lung cancer patients:
β-actin: beta actin (control); N: normal tissue; And T: cancer tissue.
7 shows the results of RT-PCR to compare the expression patterns of MAP7D2 in normal and liver cancer tissues of liver cancer patients:
β-actin: beta actin (control); N: normal tissue; And T: cancer tissue.
8 is a diagram confirming the expression of the MAP7D2 gene by RT-PCR in the kidney cancer cell line A498 knocked down by the expression of MAP7D2 by siRNA.
Figure 9 shows the results of confirming the inhibitory effect of cell growth in the kidney cancer cell line A498 knocked down expression of MAP7D2 by siRNA.
10 is a diagram confirming the expression of the MAP7D2 gene by RT-PCR in lung cancer cell line NCI-H1703 in which expression of MAP7D2 is knocked down by shRNA.
11 is a diagram observing the form of lung cancer cell line NCI-H1703 through a microscope (X100) 5 days after knocking down MAP7D2 expression (shCtrl: Nontarget shControl RNA).
12 is a graph showing changes in cell growth due to knockdown of MAP7D2 gene in lung cancer cell line NCI-H1703 (bar: standard deviation).
Figure 13 shows the change in cell migration due to knockdown of MAP7D2 gene in lung cancer cell line NCI-H1703:
A: A microscope observation of cancer cell migration experiments using transwell plates of NCI-H1703 cells transduced with shRNA (X100 magnification); And
B: Graph quantifying changes in NCI-H1703 migration due to reduced MAP7D2 expression (counting randomly selected 8-sided cell numbers at X200 magnification, bar: standard deviation).
14 is a diagram showing the change of cell invasion by knockdown of MAP7D2 gene in lung cancer cell line NCI-H1703:
A: A picture (X100 magnification) of a microscopic observation of a cancer cell infiltration experiment using a transwell plate of NCI-H1703 cells transduced with shRNA; And
B: Graph quantifying changes in NCI-H1703 infiltration due to reduced MAP7D2 expression (counting the number of cells in 8 fields randomly picked at X200 magnification, bar: standard deviation).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, containing an inhibitor of MAP7D2 (MAP7 domain containing 2) protein as an active ingredient.

The MAP7D2 protein preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

The cancer is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, stomach cancer and liver cancer, and more preferably lung cancer or kidney cancer, but is not limited thereto.

The inhibitor of expression of the MAP7D2 protein is any one selected from the group consisting of antisense nucleotides, small hairpin RNAs, small interfering RNAs, and ribozymes that complementarily bind to the mRNA of the MAP7D2 gene. Preferably, the activity inhibitor of the MAP7D2 protein is any one selected from the group consisting of compounds, peptides, peptide mimetics, matrix analogs, aptamers, and antibodies that complementarily bind to MAP7D2 protein. It is not limited.

The siRNA is composed of a 15 to 30 mer sense sequence selected from the base sequence of the mRNA of the gene encoding a human MAP7D2 protein and an antisense sequence complementary to the sense sequence, wherein the sense sequence Is not particularly limited thereto, but is preferably composed of 25 bases, but is not limited thereto. More preferably, SEQ ID NO: 4 or 5 is not limited thereto.

The antisense nucleotides, as defined in Watson-click base pairs, bind (hybridize) the complementary sequencing of DNA, immature-mRNA or mature mRNA to hinder the flow of genetic information as a protein in DNA. The nature of antisense nucleotides that are specific for the target sequence makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units they can be easily synthesized for the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as a biochemical means for studying target proteins (Rothenberg et al ., J. Natl. Cancer Inst . , ≪ / RTI > 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel type of inhibitor since there has been much progress in the field of oligonucleotide chemistry and in the field of nucleotide synthesis showing improved cell adsorption, target binding affinity and nuclease resistance.

Peptide Minetics (Peptide Minetics) is to inhibit the activity of MAP7D2 protein that inhibits the binding domain of MAP7D2 protein. Peptide mimetics can be peptides or non-peptides, and are amino acids bound by non-peptide bonds, such as psi bonds (Benkirane, N., et al. J. Biol. Chem., 271: 33218-33224, 1996). Can be configured. Furthermore, a cyclic comprising a “conformationally constrained” peptide, cyclic mimetics, at least one exocyclic domain, a binding moiety (binding amino acid) and an active site It may be a mimetics. Peptide mimetics are structured similar to the secondary structural properties of the MAP7D2 protein and are either antibodies (Park, BW et al. Nat Biotechnol 18, 194-198, 2000) or water soluble receptors (Takasaki, W. et al. Nat Biotechnol 15, 1266- 1270, 1997), which can mimic the inhibitory properties of large molecules and may be novel small molecules that can act as an equivalent to natural antagonists (Wrighton, NC et al. Nat Biotechnol 15, 1261-1265, 1997). .

The aptamer is a single-chain DNA or RNA molecule, and has a high affinity for a specific chemical or biological molecule by an evolutionary method using an oligonucleotide library called systemic evolution of ligands by exponential enrichment (SELEX). Oligomers that have the ability to select and bind can be obtained by isolation (C. Tuerand L. Gold, Science 249, 505-510, 2005; AD Ellington and JW Szostak, Nature 346, 818-822, 1990; M. Famulok, et. al., Acc. Chem. Res. 33, 591-599, 2000; DS Wilson and Szostak, Annu. Rev. Biochem. 68, 611-647, 1999). Aptamers can specifically bind to targets and modulate the activity of the targets, such as by blocking the ability of the targets to function through binding.

The antibody may specifically and directly bind to MAP7D2 to effectively inhibit the activity of MAP7D2. As the antibody that specifically binds to MAP7D2, it is preferable to use a polyclonal antibody or a monoclonal antibody. The antibody that specifically binds to MAP7D2 may be produced by a known method known to those skilled in the art, and a commercially known MAP7D2 antibody may be purchased and used. The antibody can be prepared by injecting the immunogen MAP7D2 protein into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection and can generally be administered with an adjuvant to increase antigenicity. Antibodies can be isolated by collecting blood periodically from an external host and collecting serum showing shaped titers and specificity for the antigen.

The composition may have inhibitory activity of proliferation, migration, invasion or metastasis of cancer.

In a specific embodiment of the present invention, the inventors constructed a gene expression database of a human sample through microarray analysis from gene expression data of GeneExpress Omnibus of NCBI and ArrayExpress of EBI (see Table 1), and the database contained normal And gene expression data for cancer tissues (see Table 2). Cancer Outlier Profile Analysis (COPA) was applied to discover cancer treatment targets from the cancer gene expression database built independently (Tomlins et al. 2005).

In order to select candidate target genes for cancer treatment from the constructed gene expression database, the present inventors selected genes specifically overexpressed in specific cancer tissues, and selected cell lines in which the genes were expressed higher than a certain level. In this case, the gene was selected as a novel target gene with little reported on cancer, and in this process, MAP7D2 was selected as a candidate target gene for targeted chemotherapy, and the expression of MAP7D2 was determined by microarray analysis. As a result, it was found that the tissues such as kidney cancer, lung cancer, colon cancer, ovarian cancer, stomach cancer and liver cancer were expressed higher than normal tissues or other cancer tissues (see FIG. 1). Lung cancer, colorectal cancer, ovarian cancer, gastric cancer and liver cancer cell lines were confirmed to be relatively high expression (see Figure 2). In addition, MAP7D2 gene expression was confirmed in colon cancer, lung cancer, and liver cancer cell lines that showed significantly higher MAP7D2 expression in normal tissues or other cancers, and it was confirmed that MAP7D2 gene was highly expressed in the cell line (FIG. 3 to 5). In addition, it was confirmed that MAP7D2 was expressed higher in lung cancer and liver cancer tissues than normal tissues (see FIGS. 6 and 7), and it was found that MAP7D2 was available as a target protein of colorectal cancer, lung cancer, or liver cancer.

In addition, the present inventors transduced short hairpin RNA (shRNA) or small interfering RNA (RNA) into lung or kidney cancer cell lines, which show high expression levels of MAP7D2, thereby inhibiting the expression of the MAP7D2 gene. And growth, migration, infiltration and metastasis of cancer cells of the cell line were observed. As a result, it was confirmed that MAP7D2 gene expression was inhibited by the introduction of shRNA or siRNA into cancer cells (see FIGS. 8 and 10), the proliferation of cancer cells was markedly decreased, and cell death was increased (FIGS. 9 and 11). 12), it was observed that the migration and infiltration of cancer cells are inhibited (see FIGS. 13 and 14). Therefore, it was found that inhibition of expression or activity of MAP7D2 inhibits the proliferation of cancer cells such as lung cancer or kidney cancer and plays a decisive role in invasion and metastasis.

Therefore, MAP7D2 is overexpressed in the tissues of kidney cancer, lung cancer, colorectal cancer, ovarian cancer, gastric cancer, and liver cancer and cell lines of the cancer, and inhibition of the expression of the gene inhibits the growth, migration, and invasion of cancer cells. Expression or activity inhibitors can be usefully used as an active ingredient of a pharmaceutical composition for treating cancer or inhibiting cancer metastasis.

Pharmaceutical compositions containing an inhibitor of the expression or activity of the MAP7D2 protein of the present invention as an active ingredient, preferably containing 0.0001 to 50% by weight based on the total weight of the composition, but is not limited thereto.

The pharmaceutical composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredient for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

Nucleotides or nucleic acids used in the present invention can be prepared for the purpose of oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like.

Preferably, the nucleic acid or vector is used in injectable form. Thus, in particular, the area to be treated may be mixed with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion. The pharmaceutical compositions of the present invention may in particular comprise isotonic sterile solutions or lyophilized compositions which allow the composition of injectable solutions upon the addition of dry, in particular sterile water or appropriate physiological saline. Direct injection of nucleic acid into a patient's tumor is advantageous because it allows the treatment efficiency to be focused on the infected tissue. The dosage of nucleic acid used can be adjusted by various parameters, in particular by gene, vector, mode of administration used, disease in question or alternatively desired duration of treatment. In addition, the range varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the patient. The daily dose is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times a day.

The present invention also provides a method for treating cancer or inhibiting cancer metastasis, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a subject with cancer.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. The dose may vary depending on the weight, age, sex, health condition, diet, administration period, method of administration, rate of elimination, severity of disease, and the like of a particular patient.

The pharmaceutical composition may be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection, or thoracic injection during parenteral administration. It can be administered by internal injection and can be used in the form of a general pharmaceutical formulation.

The cancer is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, stomach cancer and liver cancer, and more preferably lung cancer or kidney cancer, but is not limited thereto.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

In a specific embodiment of the present invention, the inventors have established a large-scale gene expression database of human cancer tissue to discover novel targeted anticancer candidate targets through data mining techniques. In particular, MAP7D2 is overexpressed in tissues and cells of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer, and inhibition of MAP7D2 inhibits the growth, migration, infiltration and metastasis of lung cancer or kidney cancer cells and By induction, the possibility of MAP7D2 as a target of a novel anticancer agent was confirmed. Therefore, a pharmaceutical composition containing an inhibitor of MAP7D2 protein expression or activity as an active ingredient can be usefully used for the treatment of cancer or inhibition of cancer metastasis.

In addition, the present invention provides a method for screening a candidate substance for cancer treatment or cancer metastasis inhibition using MAP7D2 protein.

The cancer is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, stomach cancer and liver cancer, and more preferably lung cancer or kidney cancer, but is not limited thereto.

Specifically,

1) treating the test substance to the cell line expressing MAP7D2 protein;

2) measuring the expression level of MAP7D2 protein in the cell line; And

3) It is preferable that the expression level of the MAP7D2 protein includes a step of selecting a test substance which is reduced compared to a control which is not treated with a test substance, but is not limited thereto.

In the method, the cell line of step 1) is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer cell lines, more preferably lung cancer or kidney cancer cell line, It is not limited to this.

In the above method, the expression level of the protein of step 2) is determined by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blotting and It is preferred to measure with any one selected from the group consisting of flow cytometry (FACS), but any method of measuring the amount of transcript or protein encoded therefrom known to those skilled in the art can be used.

Another way is,

1) treating the test substance to the MAP7D2 protein;

2) measuring the activity of the MAP7D2 protein; And

3) The activity of the MAP7D2 protein is preferably included in the step of selecting a test substance reduced compared to the control group not treated with the test substance, but is not limited thereto.

In the above method, the activity of the protein of step 2) is preferably measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry and protein chip, but is not limited thereto. .

In a specific embodiment of the present invention, a novel targeting anticancer candidate target MAP7D2 was selected through a data mining technique based on a large gene expression database of human cancer tissue constructed in the present invention, wherein the MAP7D2 is kidney cancer, lung cancer. Cancer cells that are overexpressed in tissues and cells of colon cancer, ovarian cancer, gastric cancer and liver cancer, and inhibited MAP7D2 inhibit growth, migration, invasion and metastasis, and induce apoptosis, whereby MAP7D2 plays a crucial role in cancer cells. Since it was confirmed that the method of measuring the expression or activity of the MAP7D2 protein can be usefully used for screening a substance for the treatment of cancer or inhibiting metastasis.

In addition, the present invention provides a method for monitoring or diagnosing cancer using MAP7D2 protein.

Specifically,

1) measuring the expression level of MAP7D2 protein in a subject-derived sample; And

2) preferably, but not limited to, determining a subject having an increased expression level of MAP7D2 protein as a subject at risk of cancer.

In the above method, MAP7D2 of step 1) preferably has an amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto.

In the method, the cancer of step 2) is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, stomach cancer and liver cancer, and more preferably lung cancer or kidney cancer, but not limited thereto. It doesn't work.

In a specific embodiment of the present invention, a novel targeting anticancer candidate target MAP7D2 was selected through a data mining technique based on a large gene expression database of human cancer tissue constructed in the present invention, wherein the MAP7D2 is kidney cancer, lung cancer. Cancer cells that are overexpressed in tissues and cells of colon cancer, ovarian cancer, gastric cancer and liver cancer, and inhibited MAP7D2 inhibit growth, migration, invasion and metastasis, and induce apoptosis, whereby MAP7D2 plays a crucial role in cancer cells. It was confirmed. This can be useful for monitoring or diagnosing cancer by checking the expression level of MAP7D2.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

However, the following Examples and Preparation Examples specifically illustrate the present invention, and the contents of the present invention are not limited by the Examples and Preparation Examples.

< Example  1> Database building and cancer-causing gene addiction ( oncogene addiction Looks New  Screening of Anticancer Target Genes

<1-1> Gene Expression Database of Large-scale Human Cancer Tissues

More than 450 data sets were collected and analyzed from the published gene expression databases of NCExpress' Gene Expression Omnibus and EBI's ArrayExpress, to establish a differentiated gene expression database comprising more than 33,000 samples, as shown in Table 1 below. Specifically, data was consolidated and organized in a consistent way from microarray raw data files as much as possible so that different data sets could be integrated and analyzed. First, only data using the Affymetrix human chip platform was collected. Second, the expression value at each gene level was calculated by applying Affymetrix's unique algorithm, the microarray suite 5 (MAS5) method, from the original CEL file. By applying the global mean normalization in the same way, each microarray platform (U133Plus2) allows more than 10,000 samples to be combined and meta-analyzed.

Human sample gene expression database platform U133Plus2 U133A U95A Sum data set 155 244 53 452 sample 12974 18035 2272 33,281 probe 54613 22216 12558 - Data points 708,549,062 400,647,525 28,531,776 1,137,728,363

The database contains gene expression data for at least 22,000 normal and cancerous tissues, derived from almost all tissues, as shown in Table 2 below.

group cancer normal Sum Bladder 126 23 149 Blood 1416 1127 2543 Bone Marrow 1736 5 1741 Brain 1285 2239 3524 Breast 3738 158 3896 Cervix (Cervix_ 138 46 184 Colon 1116 231 1347 Endometrium 84 81 165 Esophagus 32 32 64 Head and Neck (Head_Neck) 253 42 295 Kidney 928 166 1094 Liver 354 83 437 Lung 1290 426 1716 Lymph node 25 12 37 Lymphocyte 180 175 355 Muscle 0 622 622 Ovary 977 17 994 Pancreas 94 35 129 Prostate 473 188 661 Skin 437 80 517 Small intestine 14 7 21 Spleen 4 10 14 Stomach 295 61 356 Testis 206 26 232 Thyroid 130 52 182 Uterus 130 25 155 Etc 410 268 678 Sum 15454 6661 22115

<1-2> cancer Outlier  Profile analysis ( Cancer Outlier Profile Analysis , COPA Using) Cancer patients  Confirmation of gene expression change

As in Example <1-1>, COPA analysis was applied in a method different from the conventional approach in order to discover a therapeutic target from an independently constructed cancer gene expression database (Tomlins et al. 2005). Currently, the targeted anticancer target genes of therapeutic agents effectively used in the clinic are overexpressed by gene mutation or gene amplification in only a part of the patients. For example, Herceptin target ERBB2 is overexpressed in only about 25-30% of all breast cancer patients, and EGFR, which is the target of Iressa or Cetuximab, also affects lung cancer patients and brain tumor patients. Only about 10-20% of overexpression is observed by gene mutation or gene amplification. As such, the effective target genes are observed only in some of the patients. Therefore, as compared to the conventional t-test, the top 10 to 20% of the patients are compared to the method of finding genes that differ in expression by comparing the average of patients and normal people. Cancer outlier profile analysis (COPA) was performed because the outlier analysis method is more effective.

<1-3> New Targeting  Screening for Therapeutic Target Genes

COPA assays were applied to select genes that could be targeted for effective targeting therapy in each cancer among druggable target receptors, kinases, transporters, and channel proteins. Therapeutic drug targets currently in clinical trials or mainly on the market are mostly cell surface molecules such as receptors, channel transporters, kinases, and cell adhesion molecules (CAMs). Target genes were excavated. Candidate target genes were excavated based on the following criteria. VI) Gene expression database mining for clinical tissues formed a pool of candidate target genes that are specifically expressed in specific cancer tissues compared to normal tissues. Ii) Select genes that are specifically overexpressed in multiple cancers to become target genes in the gene pool formed, since the more specific target genes are expressed in different cancers, the more likely it is to be a direct and important gene for cancer formation. to be. Iii) Among cancer cell lines derived from the same tissues as the clinical tissues in which the target genes are expressed a lot, cell lines in which the target genes are expressed at a certain level or more were selected. Iii) A literature search was conducted on selected candidate target genes to find new addicted oncogenes by selecting genes that have not been studied as much as possible. Based on these criteria, MAP7D2 was selected and validated as the primary candidate target gene for targeted anticancer treatment among candidate target genes.

As a result, as shown in Figure 1, the expression pattern of MAP7D2 in the clinical tissue of cancer patients confirmed, kidney cancer (kidney), lung cancer (lung), colon cancer (colon), liver cancer (liver), ovary cancer (ovary) ) And the expression of MAP7D2 was increased in cancer tissues such as gastric cancer (stomach) compared to normal tissue, and it can be seen that MAP7D2 may be an effective therapeutic target for cancer treatment (FIG. 1).

< Example  2> MAP7D2 Of expression in various cancers

<2-1> in cancer cell lines Microarray  Through the platform MAP7D2  Confirmation of expression amount

Expression patterns of MAP7D2 in various cancer cell lines were analyzed using the microarray platform U133plus2A gene chip.

As a result, as shown in Figure 2, the expression pattern in the clinical tissue of MAP7D2, cancer cell lines derived from kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer in accordance with the results confirmed in the cancer tissue of Figure 1 It can be seen that the expression is relatively high in the field (Fig. 2).

In addition, based on the results of FIGS. 1 and 2, in order to select cancer cell lines to be used for experiments, cell lines that best express MAP7D2 for each target cancer selected based on the gene expression database for each cell line were identified. It was shown in Table 3 below.

Agency Cell line value Kidney cancer A498 2907 CAL54 1164 Lung cancer NCI-H1703 2588 A549 617 Colon cancer CAC02 1571 SW480 1287 Liver cancer SNU-475 1202 SNU-449 680 HepG2 643

<2-2> In Cancer Cells Reverse transcription  Polymerase chain reaction Reverse Tanscription -Polymerase Chain Reaction , RT - PCR )through MAP7D2  Confirmation of expression amount

RT-PCR was performed to confirm the expression of MAP7D2 in colorectal cancer, lung cancer and liver cancer cell lines in which MAP7D2 was overexpressed compared to other cancers.

Specifically, total RNA was isolated from 11 types of cancer cell lines (4 types of colorectal cancer, 4 types of lung cancer, and 3 types of liver cancer) using an easy-BLUE ^TM Total RNA Extraction kit (SolGent, Cat #: 17061). With 2 μg total RNA, cDNA was synthesized using DiaStar RT kit (SolGent, Cat #: DR13-R10K). cDNA was diluted with sterile distilled water to prepare 2% cDNA, followed by 10 pmole / μl Taq DNA polymerase (Solgent, Korea) and primer [Forward primer: 5'-CTCGAGAGAACAGATTATG-3 ', sequence Number 2; Reverse primer: RT-PCR was performed using 5'-CTTCACTTGTGGAGACACATC-3 ', SEQ ID NO: 3]. At this time, RT-PCR of GAPDH was performed as a gel loading control. The PCR program used was as in Table 4 below:

Temperature time Cycle 95 ℃ 2 minutes 95 ℃ 30 seconds 25 cycles
53 ℃ 30 seconds 72 ℃ 30 seconds 72 ℃ 10 minutes

As a result, as shown in Figures 3 to 5, similar to the results in Table 3, it was confirmed that the MAP7D2 gene is highly expressed in cell lines such as colorectal cancer, lung cancer and liver cancer (Fig. 3 to Fig. 5).

<2-3> in cancer tissue Reverse transcription Polymerase chain reaction  through MAP7D2  Expression level check

In order to confirm the expression pattern of MAP7D2 in lung cancer and liver cancer tissues, normal tissues and cancer tissues of lung cancer or liver cancer patients were separated, respectively, and RT-PCR was performed.

Specifically, the human resources of lung cancer and liver cancer were supplied from the Korea Human Resource Base Bank of Pusan National University Hospital. Each tissue was crushed finely using a mortar and pestle while frozen with liquid nitrogen. The pulverized tissue was isolated from total RNA using the RNeasyMini kit (Qiagene, Cat #: 74104), followed by the total RNA 2, followed by DiaStar RT kit (SolGent, Cat #: DR13-R10K). CDNA was synthesized. After diluting cDNA with sterile distilled water to prepare 2% cDNA, 10 pmole / Taq DNA polymerase (Solgent Korea) and primer [Forward primer: 5'-CTCGAGAGAACAGATTATG-3 ', SEQ ID NO: 2; Reverse primer: RT-PCR was performed using 5'-CTTCACTTGTGGAGACACATC-3 ', SEQ ID NO: 3]. At this time, RT-PCR of β-actin was performed as a gel loading control, and the PCR program used is shown in Table 4 above.

As a result, when compared with normal tissue, it was confirmed that MAP7D2 was highly expressed in lung cancer tissues of 25% or more and liver cancer tissues of 33% or more (FIGS. 6 and 7).

< Example  3> MAP7D2  Analysis of the Effect of Gene Expression Inhibition on Cancer Cell Growth

The MAP7D2 knockdown method was used to suppress the expression of the MAP7D2 gene using the MAP7D2 knockdown method using the lung cancer cell line NCI-H1703 and the renal cancer A498 cell line, which grow relatively fast among MAP7D2 and have high metastatic potential. The effect on cancer cell growth by gene expression inhibition was analyzed.

<3-1> MAP7D2  Effect of Gene Expression Inhibition on Kidney Cancer Cell Growth

To determine how MAP7D2 gene expression inhibition affects the growth of kidney cancer cells, MAP7D2 siRNA was used.

Specifically, two MAP7D2 siRNAs (# 1 Duplex; 5'-GCAGGAGAUGUUGGGAAAGAA / UUCUUUCCCAACAUCUCCUGC-3 ', SEQ ID NO: 4 and # 2: Duplex; 5'-CCCAAAUUAUUAGCUCGGAAU / AUUCCGAG, purchased from Bionia from SEQIA 5) Was used. Said siRNA was introduced into renal cancer cell line A498 through electroporation using control siRNA (Bioneer CAT #: SN_1002) and Amaxa as a control group, and 24 hours after electroporation, inhibition of MAP7D2 expression was observed. The growth of cells according to was examined in comparison with the control. In order to examine cell proliferation, first, cells of 1-4 × 10 ⁴ were dispensed into 24-well plates, incubated at 37 ° C./5% CO ₂ , and then cultured for 48 hours, respectively. The finished cells were recovered and confirmed the inhibition of MAP7D2 protein expression by MAP7D2 siRNA in renal cancer cells using the RT-PCR method described in Example 2-2.

In addition, after a predetermined time has elapsed, add 37 ul of Cell Counting Kit-8 (CCK-8, DOJINDO, Cat #: CK04-11) solution to each plate in the dark at 37 ^° C. CO ₂ incubator for 1 hour. After reaction at 100 ul was transferred to a 96 well plate each, and the degree of cell proliferation was observed by measuring the absorbance at 450nm in Victor microplate reader.

As a result, as shown in Figure 6, when treated with siRNA # 2 500nM compared to the control, it showed a clear MAP7D2 expression inhibitory effect compared to GAPDH used as an internal control (Fig. 8).

In addition, as shown in FIG. 7, when the expression of MAP7D2 siRNA was inhibited compared to the control siRNA used as a control, in the renal cancer cell line A498, after about 48 hours, about 60% of the prohibitive growth inhibition and cell death were observed. It was confirmed (Fig. 9).

<3-2> MAP7D2  Effect of Gene Expression Inhibition on Lung Cancer Cell Growth

In order to determine how MAP7D2 gene expression inhibition affects the growth of lung cancer cells, MAP7D2 shRNA was used.

Specifically, the MAP7D2 small-hairpin RNA clones in the TRC lenticiral plasmid vecotr pLKO.1-puro (Sigma) were Sigma's MISSION ^™ shRNA clones libraries (SIGMA, Cat #: SHCLNG-NM_152780). ) And used. It consists of four sets of shRNA constructs, each named TRC Nos. TRCN0000108390, TRCN0000108391, TRCN0000108392, and TRCN0000108394, respectively, and was designated as MAP7D2 shRNA # 1 to # 4. In addition to the lentiviral MAP7D2 shRNA, nontarget shRNA Control Transduction particles (SIGMA, cat #: SHC002V) were used as a negative control and MISSON TurboGFP Control Transduction as a positive control to confirm transduction efficiency and optimize shRNA delivery. particles (SIGMA, Cat #: SHC003V) were used together. Subsequently, viral particles were prepared by transfecting the shRNA into 293T using Lipofectamine ^™ 2000 (invitrogen, Cat #: 11668-027) and Enhancer Q ^™ Transfection Enhancing Reagent (WelGene, Cat #: TR001-02). After 48 hours, the virus culture supernatant was collected and filtered through a syringe filter (millipore, Cat #: SLHP033RS), and then mixed 1: 1 with fresh culture medium, in order to enhance the effect of infection, 4 μg / mL of polybrene (SIGMA, Cat #: L107689) was added and transfected into lung cancer cell lines NCI-H1703 and kidney cancer cell line A498, respectively. After 16 hours, the cells were replaced with fresh medium, incubated for 48 hours, and treated with 2 μg / ml of puromycin for 3 days to select NCI-H1703 or A498 cells transfected with MAP7D2 shRNA and selected for 3 days. . At this time, as a selection control, each cancer cell line which was not transduced with shRNA was observed by treating the same amount of puromycin. After puromycin screening, the cultured cells were recovered and the expression inhibition was confirmed using the RT-PCR method described in Example 2-2, and cell growth rates according to the inhibition of MAP7D2 expression were compared with those of the control group. The comparison was investigated.

As a result, as shown in FIG. 10, all of shMAP7D2 # 1 to # 4 showed distinct MAP7D2 expression inhibitory effect compared to β-actin used as an internal control (FIG. 10) compared to the control (FIG. 10).

11 and 12, when lung cancer cell line NCI-H1703 was transfected with four types of MAP7D2 shRNAs (# 1 to # 4), respectively, in lung cancer cell lines transfected with shMAP7D2 # 2 or # 3. Compared to the control group (Nontarget shControlRNA), cell growth was significantly reduced, and cell death was confirmed to increase (FIG. 11). In particular, it was confirmed that MAP7D2 shRNA # 2 showed about 70% growth inhibition and apoptosis effect compared to the control group for 5 days (FIG. 12).

Through this, it was confirmed that even in normal medium containing 10% serum, suppression of MAP7D2 expression lowered the dependence of growth factors and induced cell death, thereby finally lowering the extent of cell growth. Oncogene addiction is clearly shown in lung cancer, making it a good target for chemotherapy in kidney and lung cancer.

< Example  4> MAP7D2  Analysis of the Effect of Expression Inhibition on Cancer Cell Migration

In order to confirm whether the inhibition of the expression of MAP7D2 exhibits the effect of inhibiting the migration of cancer cells, a transwell (coastar, 3422) system was used and the shRNA described in Example <3-2> inhibited the expression of MAP7D2. Lung cancer cell line NCI-H1703 was used. At this time, as in the CCK-8 experiment, Nontarget shRNA Control Transduction particles were used as a negative control shRNA, and a lentiviral vector to which fluorescent material was attached was used to exclude nonspecific responses by the lentiviral vector shRNA in the cell. The efficiency was predicted. The transduction efficiency was about 80% or more. After 48 hours of transfection, 2 ug / ml of puromycin was treated for 4 days of selection, and then the cancer cells were recovered to investigate the ability of the cancer cells to migrate.

Specifically, to confirm the ability of cancer cells to migrate, try to suppress the expression of MAP7D2 with trypsin (Gibco 25300) and obtain NCI-H1703 cells screened with puromycin (RPMI, migration) (RPMI, After washing twice with 10 mM HEPES, 0.5% BSA), the cells were suspended in transfer medium at 4 × 10 ⁵ cells / ml. The 24-well transwell plate is then coated with 0.05% gelatin (Sigma G1393) on the underside of the insert for one hour at room temperature and after one hour, the gelatin remaining in the insert is removed and Wash once with PBS. After the procedure, 600 ul of RPMI transfer medium with 5% FBS was added to the chamber. After inserting the insert into the chamber using sterile tweezers, the previously prepared cells were placed in the insert at ⁴ × 10 ⁴ cells / 100 ul and incubated for 24 hours at 37 ° C./5% CO ² . To measure cell migration, the top side of the insert was first wiped with a cotton swab moistened with PBS, and the insert was placed in a chamber containing 500 ul of 3.7% paraformaldehyde and fixed at room temperature for 30 minutes. Then, 1% crystal violet (crystal violet) / 100 mM sodium borate (Sodium Borate, NaBorate) was stained with 500 ul for 30 minutes, washed with water and dried to measure the number of cells at x100 magnification under a microscope.

As a result, as shown in FIG. 11, the cells whose MAP7D2 expression was inhibited by shRNA # 2 and # 4 were markedly inhibited by about 80% compared with the non-target shRNA (shCtrl) -introduced cells. It showed an effect (Fig. 13), it can be seen that MAP7D2 plays a critical role in the migration of cancer cells.

< Example  5> MAP7D2  Analysis of the Effect of Expression Inhibition on Cancer Cell Infiltration

The effect of inhibition of expression of MAP7D2 on cancer cell invasion was analyzed.

Specifically, the cells were washed twice with RPMI invasion media (RPMI, 10 mM HEPES, 0.5% BSA) and then suspended in infiltrating medium at 4 × 10 ⁵ cells / ml. 24-well transwell plates (8 um pore size, costar 3422) were diluted in serum-free media (RPMI, 10 mM HEPES) at 1 mg / ml of Matrigel (BD 354234) at room temperature on the top of the insert. Coating for one hour. After one hour, the Matrigel remaining in the insert was removed and washed once with serum free medium. Thereafter, 600 ul of RPMI infiltration medium with 5% FBS was added. After inserting the insert into the chamber containing the medium using sterilized tweezers, 100 ul of previously prepared NCI-H1703 cells were added to the insert and incubated at 37 ° C./5% CO ² for 24 hours. To measure the infiltrated cells passing through the Matrigel, wipe the top of the insert with a cotton swab moistened with PBS, insert the insert into a chamber containing 500 ul of 3.7% paraformaldehyde (Sigma HT50) and fix at room temperature for 30 minutes. It was. Then, dye in 500 ul of 1% crystal violet (Sigma C3886) / 100 mM NaBorate (Sigma S9640) for 30 minutes, wash with water, dry, take a picture at x100 and x200 magnification under microscope and count the number of cells. It was.

As a result, as shown in FIG. 12, the cells whose MAP7D2 expression was inhibited by shRNA # 2 and # 4 were inhibited by about 70% of the marked invasion compared to the cells into which the nontarget shRNA (shCtrl) was used as a control. It showed an effect (Fig. 14), it can be seen that MAP7D2 plays a critical role in the infiltration of cancer cells.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

1. Manufacturing of powder

2 g of inhibitor of expression or activity of MAP7D2

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

MAP7D2 expression or activity inhibitor 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

MAP7D2 expression or activity inhibitor 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

10 μg / ml inhibitor of expression or activity of MAP7D2

Dilute hydrochloric acid BP until pH 7.6

Injectable sodium chloride BP up to 1 ml

MAP7D2 expression or activity was dissolved in an appropriate volume of sodium chloride BP in a suitable volume, and the pH of the resulting solution was adjusted to pH 7.6 using dilute hydrochloric acid BP, and the volume was adjusted and mixed well using a sodium chloride BP. It was. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

5. Manufacture of rings

Inhibitor of expression or activity of MAP7D2 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

6. Preparation of Granules

150 mg of inhibitor of expression or activity of MAP7D2

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

As described above, the present invention enables the development of cancer therapeutic agents using MAP7D2 expression or activity inhibitors, the development of a method for monitoring or diagnosing cancer using MAP7D2 as a biomarker, and cancer growth or metastasis. It can be usefully used to screen inhibitors.

Attach an electronic file to a sequence list

Claims (11)

delete delete delete delete delete 1) treating the test substance to the cell line expressing the MAP7D2 protein having the amino acid sequence set forth in SEQ ID NO: 1;
2) measuring the expression level of MAP7D2 protein in the cell line; And
3) screening a candidate substance for treating cancer or inhibiting cancer metastasis, comprising selecting a test substance whose expression level of the MAP7D2 protein is reduced compared to a control group not treated with the test substance.
According to claim 6, wherein the expression level of the protein of step 2) is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western Blotting And a flow cytometry (FACS). The method for screening a candidate substance for cancer treatment or cancer metastasis prevention, characterized in that it is measured by any one selected from the group consisting of.
1) treating the test substance to the MAP7D2 protein having the amino acid sequence set forth in SEQ ID NO: 1;
2) measuring the activity of the MAP7D2 protein; And
3) screening a test substance for treating cancer or inhibiting cancer metastasis, comprising selecting a test substance whose activity level of the MAP7D2 protein is reduced compared to a control which has not been treated with the test substance.
The method of claim 8, wherein the activity of the protein of step 2) is measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry and protein chip. Or a screening method for a candidate substance for inhibiting cancer metastasis.
1) measuring the expression level of MAP7D2 protein in a sample isolated from the subject in vitro; And
2) A protein detection method in vitro for providing cancer diagnosis information comprising comparing the expression level of the MAP7D2 protein of the sample of step 1) with the expression level of the MAP7D2 protein of the control sample.
The method of claim 6, 8 or 10, wherein the cancer is any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, stomach cancer and liver cancer.

Images