WO2012115437A2 - Composition pharmaceutique destinée au traitement du cancer ou à l'inhibition des métastases, contenant des inhibiteurs de l'expression ou de l'activation de la protéine map7d2, une nouvelle cible thérapeutique du cancer - Google Patents

Composition pharmaceutique destinée au traitement du cancer ou à l'inhibition des métastases, contenant des inhibiteurs de l'expression ou de l'activation de la protéine map7d2, une nouvelle cible thérapeutique du cancer Download PDF

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WO2012115437A2
WO2012115437A2 PCT/KR2012/001316 KR2012001316W WO2012115437A2 WO 2012115437 A2 WO2012115437 A2 WO 2012115437A2 KR 2012001316 W KR2012001316 W KR 2012001316W WO 2012115437 A2 WO2012115437 A2 WO 2012115437A2
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cancer
map7d2
protein
expression
metastasis
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WO2012115437A3 (fr
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권기선
김선영
최소영
도소희
박성섭
이광표
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • compositions for treating cancer or inhibiting cancer metastasis comprising inhibitors of the expression or activity of the MAP 7 D 2 protein as a novel cancer treatment target
  • the present invention relates to a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, comprising an inhibitor of expression or activity of MAP7D2 protein, which is a novel cancer growth or cancer metastasis therapeutic target.
  • Cancer Progression is Activation of Cancer-Induced Genes (oncogene) and Tumor Suppressor Genes
  • Cancer is a disease that is very heterogeneous in its cause and progression. Morphological and pathological Although similar in nature, they progress in significantly different forms at the molecular level. Recent clinical experience supports this: ERBB2 and EGFR, the targets of successful clinical drugs such as Herceptin and Zephytinib, are mutated or overexpressed in only about 20-30% of all cancer patients. have.
  • MAP7D2 (MAP7 domain containing 2) is another protein named FLJ 14503, MGC104944, RP11-393H10.2, which is a type of microtubule-associated protein 7 domain.
  • the present inventors have conducted research to discover targeted cancer treatment targets, and have established a large-scale gene expression database of cancer tissues through original data mining techniques.
  • the present invention was completed by identifying MAP7D2 that is overexpressed in lung cancer, colorectal cancer, ovarian cancer, gastric cancer, and liver cancer and remarkably inhibiting the growth, infiltration and metastasis of cancer cells by inhibition of MAP7D2 expression.
  • An object of the present invention is the MAP7D2 protein, which is a novel cancer growth or cancer metastasis therapeutic target. It is to provide a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, containing an inhibitor of expression or activity as an active ingredient.
  • Another object of the present invention is to provide a method for screening cancer growth, invasion or metastasis inhibitors using MAP7D2.
  • Another object of the present invention is to provide a method for monitoring or diagnosing cancer in a subject using MAP7D2.
  • the present invention provides a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, containing an inhibitor of the expression or activity of MAP7D2 (MAP7 domain containing 2) protein as an active ingredient.
  • MAP7D2 MAP7 domain containing 2
  • the present invention provides a method for screening a candidate substance for treating cancer or inhibiting cancer metastasis, comprising the following steps:
  • ⁇ 2i> 2 measuring the expression level of MAP7D2 protein in the cell line
  • the present invention provides a method for screening a candidate substance for cancer treatment or cancer metastasis suppression comprising the following steps:
  • the present invention provides a method for monitoring or diagnosing cancer using MAP7D2 protein:
  • the present invention also provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of an MAP7D2 protein expression or activity inhibitor.
  • the present invention also provides a method for treating cancer or inhibiting cancer metastasis, comprising administering a pharmaceutically effective amount of an MAP7D2 protein expression or active inhibitor to a subject with cancer. Provide the law.
  • the present invention comprises the step of measuring the expression level of MAP7D2 in cancer cells using any one or more of the nucleic acid complementary to the antibody or gene specifically binding to MAP7D2, the diagnosis of cancer, confirming the treatment result Or provide a method of assessing prognosis.
  • the present invention also provides a kit for diagnosing cancer, comprising any one or more of nucleic acids complementary to antibodies or / genes that specifically bind to MAP7D2.
  • the present invention also provides an inhibitor of MAP7D2 protein expression or activity for use in the prophylaxis or treatment of cancer or the pharmaceutical composition for inhibiting cancer metastasis.
  • the present invention provides nucleic acids complementary to antibodies or genes that specifically bind to MAP7D2 for use in cancer diagnostic kits.
  • a large-scale gene expression database of human cancer tissue was constructed to discover a novel targeting anticancer candidate target MAP7D2 through data mining techniques, and the MAP7D2 is a kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer, and the like. It is particularly remarkably overexpressed in tissue or cells of liver cancer, and inhibition of MAP7D2 significantly inhibits the growth, migration, invasion and metastasis of cancer cells and induces apoptosis, thereby inhibiting the expression or activity inhibitor of MAP7D2 protein.
  • a pharmaceutical composition containing as an ingredient may be usefully used as an active ingredient of a pharmaceutical composition for treating cancer or inhibiting cancer metastasis.
  • FIG. 1 is a diagram showing the results of analyzing the expression pattern of the MAP7D2 gene in clinical tissues with 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.
  • FIG. 3 illustrates the use of GAPDH as an internal control for standardization of RNA amounts.
  • Figure shows the expression of MAP7D2 in colorectal cancer cell line.
  • Figure shows the expression of MAP7D2 in liver cancer cell line.
  • FIG. 5 uses GAPDH as an internal control for standardization of RNA amounts.
  • FIG. 6 shows the results of RT-PCR to compare the expression of MAP7D2 in normal and lung cancer tissues of lung cancer patients:
  • -Act in beta actin (control); N: normal tissue; And T: cancer tissue.
  • FIG. 7 shows the results of RT-PCR to compare the expression patterns of MAP7D2 in normal and liver cancer tissues of liver cancer patients:
  • ⁇ -act in beta actin (control); N: normal tissue; And T: cancer tissue.
  • FIG. 8 shows the renal cancer cell line A498 knocked down by the expression of MAP7D2 by siRNA.
  • MAP7D2 gene was confirmed by RT-PCR.
  • Figure 9 shows the results of confirming the inhibitory effect of cell growth in the renal cancer cell line A498 knocked down expression of MAP7D2 by siRNA.
  • FIG. 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.
  • FIG. 11 is a view of morphology of lung cancer cell line NCI-H1703 observed under a microscope (X100) 5 days after knocking down MAP7D2 expression (shCtrl: Nontarget shControl RNA).
  • FIG. 12 is a graph showing changes in cell growth due to knockdown of the MAP7D2 gene in lung cancer cell line NCI-H1703 (bar: standard deviation).
  • FIG. 13 is a diagram showing the change in cell migration by knockdown of MAP7D2 gene in lung cancer cell line NCI-H1703:
  • A A picture of a microscopic observation of a cancer cell migration experiment using a transwell plate of NCI-H1703 cells transduced with shRNA (X100 magnification);
  • FIG. 14 is a diagram showing the change of cell invasion by knockdown of MAP7D2 gene in lung cancer cell line NCI-H1703:
  • the present invention provides a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, comprising an MAP7D2 (MAP7 domain containing 2) protein expression or activity inhibitor as an active ingredient.
  • MAP7D2 MAP7 domain containing 2
  • 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, gastric 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 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.
  • the MAP7D2 protein inhibitor is any one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that complementarily bind to MAP7D2 proteins. Is preferably, but is not limited thereto.
  • 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, and more preferably, SEQ ID NO: 4 or 5, but is not limited thereto.
  • the antisense nucleotide binds to (generalizes) the complementary sequencing of DNA, immature -mRNA, or mature mRNA, as defined by the Watson-click base pair, thereby disrupting the flow of genetic information as a protein in DNA. will be.
  • the nature of antisense nucleotides that are specific for the target sequence makes them exceptionally versatile. 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 biochemical means for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81: 1539-1544, 1999).
  • antisense nucleotides can be considered as a new type of inhibitor because of recent advances in oligonucleotide chemistry and in the synthesis of nucleotides that exhibit improved cell adsorption, target binding affinity and nuclease resistance.
  • Peptide Minetics binds to the binding domain of the MAP7D2 protein. It is to inhibit the activity of the inhibitory MAP7D2 protein.
  • Peptide or non-systematic seuneun peptide may be a non-peptide, S p i coupling (Benkirane, N., et al J. Biol Chem, 271:... 33218-33224, 1996) and engaged by the same, a non-peptide bond Can be composed of amino acids.
  • a "conformational ly constrained" peptide, between cyclic mimetics, at least one exocyclic domain, binding moiety (binding amino acid) and active site It may be a click 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 work with the same effect as natural antagonists (Wrighton, NC et al. Nat Biotechnol 15, 1261-1265, 1997).
  • the aptamer is a single chain DNA or RNA molecule
  • Oligomers that bind with specific affinity and selectivity to specific chemical or biological molecules by evolutionary methods using oligonucleotide libraries called systemat ic evo 1 on i 1 and g of s by exponential enrichment (SELEX) Can be obtained separately (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 MAP7D2 activity.
  • MAP7D2 it is preferable to use a polyclonal antibody or a monoclonal antibody.
  • the antibody that specifically binds to MAP7D2 may be prepared by known methods known to those skilled in the art, and commercially known MAP7D2 antibodies 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 intramuscularly, intraperitoneally or subcutaneously, and can generally be administered in combination with an adjuvant to increase antigenicity. Blood is drawn periodically from an external host to determine the titer and antigen Antibodies can be isolated by collecting specific serum.
  • the composition may have inhibitory activity of cancer proliferation, migration, invasion or metastasis.
  • the present inventors describe the gene expression of NCBI.
  • the present inventors selected genes that are specifically overexpressed in specific cancer tissues, and selected cell lines in which the genes were expressed higher than a certain level. At this time, the gene was selected as a novel target gene with little reported on cancer. In this process, MAP7D2 was selected as a candidate target gene for targeted chemotherapy, and the expression of MAP7D2 was analyzed by microarray analysis. As a result, it was found that the renal cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer were expressed higher than normal tissues or other cancer tissues (see FIG. 1).
  • the present inventors transduced short hairpin RNA (shRNA) or small interfering RNA (LNA) genes into lung or kidney cancer cell lines, which show high expression levels of MAP7D2. Expression was inhibited 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 introduction of shRNA or siRNA into cancer cells (see FIGS. 8 and 10), and proliferation of cancer cells was markedly reduced and cell death was increased. 9 and 11 to 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.
  • shRNA short hairpin RNA
  • LNA small interfering RNA
  • MAP7D2 is overexpressed in the tissues of renal cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer and the cell lines of the cancer, and the inhibition of the expression of the gene inhibits the growth, migration and invasion of cancer cells.
  • Inhibitors of the expression or activity of MAP7D2 protein may be usefully used as an active ingredient of a pharmaceutical composition for treating cancer or inhibiting cancer metastasis.
  • the pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • Pharmaceutically acceptable carriers include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and one or more of these components in combination.
  • Other conventional additives such as antioxidants, buffers, bacteriostatics, can be added.
  • diluents, dispersants, surfactants, binders and lubricants can be added in addition to formulate into injectable formulations, such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, which will act specifically on target organs.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, which will act specifically on target organs.
  • Target organ specific antibodies or other ligands can be used in combination with the carrier so that they can be used.
  • Remington's Pharmaceutical Science Remington's Pharmaceutical Science (Recent), Mack Publishing Company, Easton PA, it is preferred according to each disease or component. It may be formulated.
  • the nucleotides or nucleic acids used in the present invention may be prepared for the purpose of oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like.
  • the nucleic acid or vector is used in injectable form.
  • the area to be treated may be mixed with any pharmaceutically acceptable media 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.
  • nucleic acid used in tumors of the 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 required duration of treatment. In addition, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and the severity of the disease.
  • the daily dose is about 0.0001 to 100 /, preferably 0.001 to 10 /, preferably administered once to several times a day.
  • 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, gastric cancer and liver cancer, and more preferably lung cancer or kidney cancer, but is not limited thereto.
  • 3) preferably, but not limited to, selecting a test substance whose expression level of the MAP7D2 protein is reduced compared to a control group not treated with the test substance.
  • 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, and the lung cancer or kidney cancer cell lines. More preferably, it is not limited to this.
  • the expression level of the protein of step 2) is immunoprecipitation method.
  • the activity level of the MAP7D2 protein is compared to the control group not treated with the test substance. It is preferable to include the step of screening a reduced test substance compared to but is not limited thereto.
  • the activity level of the protein of step 2) is SDS-PAGE ,.
  • Method, enzyme immunoassay (ELISA), mass spectrometry and protein chip is preferably measured by any one selected from the group consisting of, but not limited to.
  • 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. Cancer cells that are overexpressed in tissues and cells of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer, and inhibited MAP7D2 inhibit growth, migration, invasion and metastasis, and induce apoptosis. Since it has been determined to play a crucial role in the method, the method of measuring the expression or activity of MAP7D2 protein can be usefully used for screening a substance for inhibiting cancer treatment or cancer metastasis.
  • the present invention provides a method for monitoring or diagnosing cancer using MAP7D2 protein.
  • MAP7D2 in step 1) preferably has an amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto.
  • 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. Or not limited thereto.
  • the present invention provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of an MAP7D2 protein expression or activity inhibitor.
  • the present invention provides a method for treating cancer or inhibiting cancer metastasis, comprising administering a pharmaceutically effective amount of an MAP7D2 protein expression or active inhibitor to a subject with cancer. Provide the law.
  • the pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably
  • the dosage can be varied according to the body of the specific patient, age, sex, health status, 'diet, administration time, administration method, clearance, such as the severity of the disease.
  • the MAP7D2 protein expression or activity inhibitor may be administered orally or orally during clinical administration and intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, intrauterine It can be administered by injection, cerebrovascular injection, or intrathoracic injection, and can be used in the form of general pharmaceutical preparations.
  • the cancer is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric 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, or cat, and most preferably anthropoid animals such as chimpanzees or gorillas. to be.
  • an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, or cat
  • anthropoid animals such as chimpanzees or gorillas. to be.
  • the present inventors constructed a large-scale gene expression database of human cancer tissue to find a novel targeted anticancer candidate target through data mining techniques.
  • 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, invasion and metastasis of lung cancer or kidney cancer cells and apoptosis.
  • This induction confirmed the possibility of MAP7D2 as a target of a novel anticancer agent, and therefore inhibitors of the expression or activity of MAP7D2 protein can be usefully used for the treatment of cancer or inhibition of cancer metastasis.
  • the present invention includes the steps of measuring the expression level of MAP7D2 in cancer cells using at least one of an antibody specifically binding to MAP7D2 or a nucleic acid complementary to the gene, the result of the diagnosis and treatment of cancer It provides a method of evaluating or evaluating prognosis.
  • the cancer is preferably any one selected from the group consisting of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer, and liver cancer, and more preferably lung cancer or kidney cancer, but is not limited thereto.
  • detection of expression of MAP7D2 elevated above normal It means that the patient has cancer.
  • detection of normal expression of MAP7D2 in diagnostic samples of individuals treated or receiving cancer means that the cancer treatment is successful, and detection of elevated MAP7D2 above normal in the diagnostic samples indicates that treatment should be continued. do.
  • detection of normal expression of MAP7D2 in a diagnostic sample of a subject with cancer means a good prognosis, and detection of MAP7D2 elevated above normal in the diagnostic sample means a poor prognosis.
  • the present invention provides a kit for diagnosing cancer comprising any one or more of nucleic acids complementary to an antibody or gene that specifically binds to MAP7D2.
  • the cancer diagnostic kit may further include one or more substances and reaction product detection reagents and instructions for reacting with MAP7D2.
  • the one or more substances that react with MAP7D2 may be RNA or DNA complementary to the RNA or DNA of MAP7D2, and an antibody that binds to the MAP7D2 protein and the reagent for detecting the reaction product may be a nucleic acid or protein label and a coloring reagent. have.
  • 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. Cancer cells that are overexpressed in tissues and cells of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer and liver cancer, and inhibited MAP7D2 inhibit growth growth, invasion and metastasis, and induce apoptosis, thereby inducing MAP7D2 to cancer cells. It was confirmed to play a decisive role. Therefore, the expression level of MAP7D2 can be monitored and cancer can be diagnosed.
  • the present invention is for use in the prevention or treatment of cancer or to inhibit cancer metastasis
  • Inhibitors of MAP7D2 protein expression or activity are provided.
  • the MAP7D2 protein preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.
  • the inhibitor of expression of the MAP7D2 protein may be any one selected from the group consisting of antisense nucleotides, short interfering RNAs, and short hairpin RNAs, which complementarily bind to mRNA of a gene. Preferred but not limited to.
  • the inhibitor of activity of the MAP7D2 protein binds complementarily to the MAP7D2 protein. It is preferably one selected from the group consisting of a compound, a peptide, a peptide mimetics, an aptamer and an antibody, 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, gastric cancer, and liver cancer, and more preferably lung cancer or kidney cancer, but is not limited thereto.
  • the inventors constructed a large-scale gene expression database of human cancer tissue to discover novel targeted anticancer candidate targets through data mining techniques.
  • 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 apoptosis. Because of this induction, inhibitors of the expression or activity of the MAP7D2 protein can be usefully used for preventing or treating cancer or inhibiting cancer metastasis.
  • the present invention provides a nucleic acid complementary to an antibody or gene that specifically binds to MAP7D2 for use in a kit for cancer diagnosis.
  • the cancer diagnostic kit may further include one or more substances and reaction products for detecting the reaction with MAP7D2 and instructions thereof.
  • the one or more substances that react with MAP7D2 may be RNA or DNA complementary to the RNA or DNA of MAP7D2, and an antibody that binds to MAP7D2 protein
  • the reagent for detecting the reaction product may be a nucleic acid or protein label and a coloring reagent.
  • 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. Cancer cells that are overexpressed in tissues and cells of kidney cancer, lung cancer, colon cancer, ovarian cancer, gastric cancer, and liver cancer, and inhibit MAP7D2 growth, migration, invasion and metastasis, and induce apoptosis, thereby inhibiting MAP7D2 from cancer cells. It was confirmed that it plays a decisive role in. Thus, nucleic acids complementary to antibodies or / genes that specifically bind to MAP7D2 can be usefully used in cancer diagnostic kits.
  • the database contains gene expression data for more than 22,000 normal and cancerous tissues from almost all tissues, as shown in Table 2 below.
  • COPA Cancer Outlier Profile Analysis
  • Example ⁇ 1-1> C0PA analysis was applied in a method different from the conventional approach to discover therapeutic targets from a cancer gene expression database constructed independently (Tomlins et al. 2005). .
  • 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.
  • ERBB2 a target of Herceptin
  • EGFR a target of Iressa or Celuximab
  • Overexpression by gene mutation or gene amplification is observed only in about 10-20% of the population.
  • the outlier analysis method compares the average of the top 10-20% of the patient group with the average of the normal person, rather than comparing the average of the patient group and the normal person to find genes with different expressions as in the conventional t-test. Since it is effective, this cancer outlier profile analysis (COPA) was performed.
  • COPA cancer outlier profile analysis
  • the C0PA assay was applied to select genes that could be targeted for effective targeting therapy in each cancer from among drug-targeting 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).
  • Candidate target genes were identified.
  • Candidate target genes were identified based on the following criteria. i) Gene expression database mining for clinical tissues formed a pool of candidate target genes that are expressed more specifically in specific cancer tissues than in normal tissues. ii) Select genes that are specifically overexpressed in several cancers to become target genes in the gene pool formed.
  • MAP7D2 As a result, as shown in FIG. 1, as a result of confirming the expression pattern of MAP7D2 in the clinical tissues of cancer patients, kidney cancer, lung cancer, colon cancer, liver cancer, and ovary It was observed that the expression of MAP7D2 was increased in cancer tissues such as cancer and stomach cancer compared to normal tissues, and thus, MAP7D27 ⁇ could be an effective therapeutic target for the treatment of cancer (FIG. 1). ).
  • the expression pattern in the clinical tissue of MAP7D2 is 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 they are relatively high in one cancer cell line (FIG. 2).
  • MAP7D2 is best expressed for each target cancer selected based on the gene expression database for each cell line, in order to select cancer cell lines to be used in the experiment. The cell lines were confirmed, which are shown in Table 3 below.
  • RT-PCR was performed to confirm the expression patterns of MAP7D2, respectively.
  • 11 types of cancer cell lines (4 types of colorectal cancer, 4 types of lung cancer, and 3 types of liver cancer) were used in the easy-BLUE TM Total RNA Extraction kit (Sol Gent, Cat #: 17061).
  • cDNA was synthesized using a DiaStar RT kit (SolGent, Cat #: DR13-R10K) with each total RNA 2 / g. cDNA was distilled with sterile distilled water to prepare 2% cDNA, followed by 10 pmole Taq DNA polymerase (Solgent Korea) and primer [Forward primer: 5 '-CTCGAGAGAACAGATTATG-3', sequence No. 2; Reverse primer: RT-PCR was performed using 5'-CTCACTTGTGGAGACACATC-3 ', SEQ ID NO: 3]. At this time, as a gel loading control RT-PCR of GAPDH was performed. The PCR programs used were as shown in Table 4 below.
  • MAP7D2 gene is highly expressed in cell lines such as colorectal cancer, lung cancer and liver cancer as shown in Table 3 above ( 3 to 5).
  • RNA 2 and the DiaStar RT kit 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. Using the RNeasyMini kit (Qiagene, Cat #: 74104), the pulverized tissue was isolated from the total RNA, and then the total amount of RNA 2 and the DiaStar RT kit (SolGent, Cat #: DR13-R10K). CDNA was synthesized.
  • cDNA was distilled with sterile distilled water to prepare 2% cDNA, followed by 10 pmole / Taq DNA polymerase (Solgent, Korea) and primer primer: 5 '-CTCGAGAGAACAGAGATTATG-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 ⁇ -act in was performed as a gel loading control, and the PCR program used is shown in Table 4 above.
  • MAP7D2 was highly expressed in lung cancer tissues and more than 33% in liver cancer tissues when compared with normal tissues (FIGS. 6 and 7).
  • the knockdown method was used to inhibit MAP7D2 gene expression and to analyze the effect on cancer cell growth by MAP7D2 gene expression inhibition.
  • MAP7D2 siRNA was used to determine how MAP7D2 gene expression inhibition affects the growth of kidney cancer cells.
  • each plate has a Cell Counting Kit-
  • the degree of cell proliferation was observed by measuring absorbance at 450 nm in Victor microplate reader.
  • MAP7D2 shRNA was used to cut.
  • Virus particles were prepared by transfecting the shRNA into 293T using Transfection Enhancing Reagent (WelGene, Cat #: TR001-02). After 48 hours, the virus culture supernatant was recovered and filtered with syringe filter (mi 11 ipore, Cat #: SLHP033RS), and then mixed 1: 1 with fresh culture medium, in order to improve the effect of infection. 4 yg / niL polybrene (SIGMA, Cat #: L107689) was added and transfected into lung cancer cell lines NCI-H1703 and kidney cancer cell line A498, respectively.
  • Transfection Enhancing Reagent WelGene, Cat #: TR001-02
  • syringe filter mi 11 ipore, Cat #: SLHP033RS
  • 4 yg / niL polybrene SIGMA, Cat #: L107689 was added and transfected into lung cancer cell lines NCI-H1703 and kidney cancer cell line A498, respectively.
  • MAP7D2 shRNA # 2 or # 3 was transfected.
  • cell growth was markedly decreased and cell death was increased compared to the control group (Nontarget shControlRNA) (FIG. 11).
  • MAP7D2 shRNA # 2 was confirmed to exhibit about 70% growth inhibition and apoptosis effect compared to the control group (FIG. 12).
  • MAP7D2 has a clear oncogene addiction in renal and lung cancers, and thus may be a good target for chemotherapy in renal and lung cancers.
  • Transwell (transwell Kcoastar, 3422) system was used to confirm whether the inhibition of MAP7D2 expression by cancer cells, the expression of MAP7D2 by the shRNA described in Example ⁇ 3-2> Lung cancer cell line NCI-H1703 was inhibited.
  • Nontarget shRNA Control Transduction particles were used as negative control shRNA to exclude nonspecific reaction by lentiviral vector shRNA in cells, and the lentiviral vector to which fluorescent material was attached was used.
  • the introduction efficiency was predicted. The efficiency of introduction was about 80% or more. After 48 hours of phenotypic infection, 2 ug / ml of puromycin was used for 4 days of selection, and then the cancer cells were recovered and examined for their ability to migrate.
  • the cells were suspended in transfer medium at 4 ⁇ 10 cells / ml.
  • the 24-well transwell plate is then coated with 0.05% gelatin (Sigma G1393) on the underside of the insert (i nser t) for one hour at room temperature and after one hour, the remaining gelatin in the insert.
  • 600 ul of RPMI transfer medium with 53 ⁇ 4 FBS was added to the chamber.
  • the cells were prepared in advance, the cells were placed in the insert into 4xl0 4 year old catcher / 100 ul and incubated for 24 hours at 37 ° C / 5% CO 2 conditions.
  • the cells whose MAP7D2 expression was suppressed by shRNA # 2 and # 4 were about 80% compared with the cells into which the nontarget shRNA (shCtr 1) introduced as a control was introduced. It showed a clear movement inhibitory effect (Fig. 13), it can be seen that MAP7D2 plays a critical role in the migration of cancer cells.
  • the cells were washed twice with RPMI invasion media (RPMI, lOmM HEPES, 0.5% BSA), and then the cells were suspended in the infiltration medium at 4 ⁇ 10 cells / ml.
  • 24-well transwell plates (8 urn pore size, costar 3422) were diluted with Matrigel (BD 354234) in serum-free media (RPMI, lOmM HEPES) at 1 mg / ml at room temperature on the top of the insert. Coating for one hour at. After one hour, the Matrigel remaining in the insert was removed and washed once with serum-free medium. Then, 600 ul RPMI infiltration medium with 5% FBS was added.
  • RPMI invasion media RPMI, lOmM HEPES, 0.5% BSA
  • the cells whose MAP7D2 expression was suppressed by shRNA # 2 and # 4 were about 70% compared with the cells into which the nontarget shRNA (shCtr 1) introduced as a control was introduced. It showed a significant degree of invasion inhibitory effect (FIG. 14), and it can be seen that MAP7D2 plays a critical role in cancer cell invasion.
  • the above ingredients were mixed and layered in an airtight cloth to prepare a powder.
  • tablets were prepared by tableting according to a conventional method for preparing tablets.
  • the capsule was prepared by filling in gelatin capsules according to a conventional method for preparing capsules.
  • the solution was layered in a 5 mi type I ampoule made of clear glass, encapsulated under the upper grid of air by dissolving the glass, and sterilized by autoclaving at 12 C C for at least 15 minutes to prepare an injection solution.
  • the present invention enables the development of cancer therapeutic agents using MAP7D2 expression or activity inhibitors, and the development of a method for monitoring or diagnosing cancer using MAP7D2 as a biomarker. It may be usefully used for screening cancer growth or metastasis inhibitors.

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Abstract

La présente invention concerne une composition pharmaceutique destinée au traitement du cancer ou à l'inhibition des métastases, contenant des inhibiteurs de l'expression ou de l'activation de la protéine MAP7D2 en tant que principes actifs. Plus spécifiquement, une nouvelle cible thérapeutique anticancéreuse a été découverte au moyen de techniques d'exploration de données, par la construction d'une base de données d'expression génétique à grande échelle de tissus cancéreux humains ; sur la base des profils de l'expression génétique de lignées cellulaires cancéreuses, des lignées cellulaires cancéreuses appropriées ont été choisies et vérifiées expérimentalement par l'inhibition de gènes cibles candidats. Ceci a permis de confirmer que la MAP7D2 constituait une nouvelle cible anticancéreuse potentielle, qui est surexprimée dans toute une série de cellules ou de tissus cancéreux ‑ notamment les cellules ou tissus cancéreux du rein, du poumon, du gros intestin, de l'ovaire, de l'estomac et du foie ‑ et dont l'inhibition empêche la croissance, le mouvement, l'infiltration et la métastase de cellules cancéreuses, provoquant ainsi la destruction cellulaire. Ainsi, les niveaux d'expression de MAP7D2 peuvent être utiles en tant que marqueurs diagnostiques du cancer, et un inhibiteur d'activation ou d'expression de la protéine MAP7D2 peut être utilisé en tant que principe actif dans une composition pharmaceutique destinée au traitement du cancer ou à l'inhibition des métastases.
PCT/KR2012/001316 2011-02-25 2012-02-21 Composition pharmaceutique destinée au traitement du cancer ou à l'inhibition des métastases, contenant des inhibiteurs de l'expression ou de l'activation de la protéine map7d2, une nouvelle cible thérapeutique du cancer Ceased WO2012115437A2 (fr)

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KR1020120016917A KR101376599B1 (ko) 2011-02-25 2012-02-20 신규한 암 치료 표적인 ｍａｐ７ｄ２ 단백질의 발현 또는 활성 억제제를 포함하는 암 치료 또는 암 전이 억제용 약학적 조성물

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