WO2021075662A1 - Composition de biomarqueur comprenant mir-320c en tant que principe actif pour la prédiction de la réactivité à un médicament d'un patient atteint d'un cancer du sein triple négatif à un agent anticancéreux à base de platine - Google Patents

Composition de biomarqueur comprenant mir-320c en tant que principe actif pour la prédiction de la réactivité à un médicament d'un patient atteint d'un cancer du sein triple négatif à un agent anticancéreux à base de platine Download PDF

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WO2021075662A1
WO2021075662A1 PCT/KR2020/007605 KR2020007605W WO2021075662A1 WO 2021075662 A1 WO2021075662 A1 WO 2021075662A1 KR 2020007605 W KR2020007605 W KR 2020007605W WO 2021075662 A1 WO2021075662 A1 WO 2021075662A1
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mir
platinum
breast cancer
triple negative
negative breast
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Korean (ko)
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박종훈
임세라
김예솔
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Geninus Inc
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Geninus Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • TNBC Triple Negative Breast Cancer
  • ER estrogen receptor
  • PR progesterone receptor
  • HER2 human epidermal growth factor receptor
  • TNBC patients are aggressive and have the worst prognosis.
  • Luminal A, Luminal B and HER2 Unlike other breast cancer subtypes, TNBC treatment is particularly difficult because there are no three hormone receptors as therapeutic targets.
  • MicroRNAs are small non-coding RNAs, which bind to the 3'-untranslated regions (UTRs) of target mRNAs and regulate target gene expression. Numerous studies have shown that miRNAs are important for biological processes including cell proliferation, cell death, development, signal transduction, and their association with cancer. In addition, it has been reported in several studies that miRNA affects drug responsiveness and resistance by regulating target gene expression in breast cancer and non-small cell lung cancer. According to previous studies, the miR-320 family has been associated with the reprogramming of the tumor microenvironment and characteristics of various cancers in ovarian, cervical and gastric cancers. In addition, miR-320c has been reported to be related to gemcitabine-resistance in pancreatic cancer through SMARCC1 and to tumor behavior in bladder cancer through CDK6 regulation.
  • the survival of cancer cells is dependent on increased levels of stress, such as the degree of remission of DNA damage induced during the tumorigenic process and increased replication stress. Therefore, inhibition of the stress reduction pathway and application of stress overload are expected to increase the stress level to a critical level specifically in tumor cells.
  • chemotherapy targeting DNA repair (platinum compounds) or p53 (taxane) is used in many cases in TNBC patients.
  • platinum-based compound the clinical activities of cisplatin and carboplatin on breast cancer have been reported several times, but the clinical effect on TNBC tumors is insufficient. Therefore, a new combination therapy strategy for TNBC patients is needed.
  • Oxaliplatin is one of the platinum-based compounds and does not cross-react with cisplatin and carboplatin.
  • oxaliplatin has shown anti-tumor activity in ovarian cancer, non-small cell lung cancer and breast cancer.
  • TNBC due to the heterogeneity of TNBC, it is still difficult to predict a treatment prognosis for TNBC and to establish a treatment strategy.
  • An object of the present invention is to provide a biomarker composition for predicting reactivity to platinum-based anticancer drugs in triple negative breast cancer patients comprising miR-320c as an active ingredient.
  • another object of the present invention is a composition for predicting the reactivity to a platinum-based anticancer agent in a triple negative breast cancer patient comprising an agent capable of measuring the expression level of miR-320c as an active ingredient, or a platinum in a triple negative breast cancer patient comprising the same It is to provide a kit for predicting the reactivity of anticancer drugs.
  • another object of the present invention is a method of providing information for predicting the reactivity of a triple negative breast cancer patient to a platinum-based anticancer agent comprising measuring the expression level of miR-320c from a sample isolated from a triple negative breast cancer patient It is to provide.
  • another object of the present invention is to provide a pharmaceutical composition or a health functional food composition for enhancing the anticancer effect of a platinum-based anticancer agent in a triple negative breast cancer patient comprising a miR-320c, miR-320c expression promoter or an activator as an active ingredient.
  • Another object of the present invention is a miR-320c, miR-320c expression promoter or activator; And it is to provide a pharmaceutical composition for preventing or treating triple negative breast cancer comprising a platinum-based anticancer agent as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating triple negative breast cancer in patients with increased expression of miR-320c comprising a platinum-based anticancer agent as an active ingredient.
  • Another object of the present invention is to provide a method for screening a sensitivity enhancer for platinum-based anticancer agents by measuring the expression level of miR-320c.
  • another object of the present invention is to provide a triple negative breast cancer treatment method comprising the step of selecting a triple negative breast cancer patient with an increased expression level of miR-320c and administering a platinum-based anticancer agent.
  • the present invention provides a composition for predicting the reactivity of a platinum-based anticancer agent in patients with triple negative breast cancer, comprising an agent capable of measuring the expression level of miR-320c as an active ingredient.
  • the present invention provides a kit for predicting reactivity to platinum-based anticancer drugs in patients with triple negative breast cancer comprising the composition.
  • the present invention comprises the steps of: (1) measuring the expression level of miR-320c from a sample isolated from a triple negative breast cancer patient; (2) comparing the measured expression level of miR-320c with a control sample; And (3) determining that the measured expression level of miR-320c is higher than that of the control sample, determining that it has sensitivity to platinum-based anticancer drugs. Provides a way to provide.
  • the present invention provides a pharmaceutical composition for enhancing the anticancer effect of a platinum-based anticancer agent in a triple negative breast cancer patient comprising a miR-320c, miR-320c expression promoter or an activator as an active ingredient.
  • the present invention provides a health functional food composition for enhancing anticancer effect against platinum-based anticancer agents in triple negative breast cancer patients comprising a miR-320c, miR-320c expression promoter or activator as an active ingredient.
  • the present invention miR-320c, miR-320c expression promoter or activator; And it provides a pharmaceutical composition for preventing or treating triple negative breast cancer comprising a platinum-based anticancer agent as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating triple negative breast cancer in patients with increased expression of miR-320c comprising a platinum-based anticancer agent as an active ingredient.
  • the present invention comprises the steps of selecting a patient with triple negative breast cancer with an increased expression level of miR-320c; And administering a platinum-based anticancer agent to the selected triple negative breast cancer patient.
  • the present invention relates to a biomarker composition for predicting drug responsiveness of patients with triple negative breast cancer to a platinum-based anticancer drug containing miR-320c as an active ingredient.
  • CHEK1 expression was directly regulated by miR-320c, which was down-regulated in most TNBC cell lines.
  • the present inventors have shown that the ectopic restoration of miR-320c in oxaliplatin-treated TNBC cells is through negative regulation of CHEK1, inhibiting DNA damage response, DNA repair, and improving genomic instability, and It has been found to activate death.
  • the combination of miR-320c mimic and oxaliplatin effectively inhibited tumor progression.
  • miR-320c plays an important role in the reactivity of TNBC cells to oxaliplatin by regulating CHEK1 expression, which is a therapeutic target for increasing chemotherapy responsiveness, which will be usefully utilized in triple-negative breast cancer treatment strategies. It is expected to be able to.
  • CHEK1 mRNA expression measured through qRT-PCR is shown. h18s rRNA was used as an endogenous control.
  • E The results of Western blot analysis of Chk1 in breast cancer cell lines are shown. ⁇ -actin was used as a loading control.
  • F The sequence alignment result of the 3'UTR of CHEK1 containing miR-320c and two binding sites is shown. WT1 and WT2; Wild type CHEK1 3'UTR seed sequence, MT1 and MT2; Mutant CHEK1 3'UTR seed sequence.
  • the mutated sequence is indicated by white letters.
  • G After transfection with miR-320c mimic (miR-320c) or negative control mimic (NC), it shows luciferase activity of CHEK1 wild type (WT) or mutant 3'UTR construct (MT) in HEK293T cells. . NS , no significance.
  • H After transfection with negative control (NC) or miR-320c (320c) for 48 hours, TNBC cell lines (MDA-MB-231, HS578T, MDA-MB-468) showing miR-320c transfection efficiency This is the result of qRT-PCR analysis.
  • IJ It is shown that miR-320c overexpression in TNBC cell lines (MDA-MB-231, HS578T, MDA-MB-468) decreases CHEK1 mRNA and protein expression levels.
  • FIG. 3 shows the results that upregulation of miR-320c increases apoptosis through regulation of Chk1 expression.
  • A-B It shows the results of flow cytometry of apoptotic cells.
  • C After transfection with negative control mimic (NC) or miR-320c mimic (miR-320c) and after treatment with oxaliplatin (50 ⁇ M) for 72 hours, miR-320c transfection in MDA-MB-23 cells It is the result of qRT-PCR analysis showing infection efficiency.
  • D-E The results of treatment of MDA-MB-231 transfected with miR-320c mimic with oxaliplatin (oxa) for 72 hours are shown.
  • F CHEK1 siRNA shows the result of decreasing the expression level of Chk1 protein.
  • G-H It shows the results of flow cytometry of apoptotic cells.
  • FIG. 4 shows the result that the increase in miR-320c induces the accumulation of DNA damage through Chk1 regulation.
  • A A representative image of MDA-MB-231 is shown after transfection of a negative control mimic (NC) or miR-320c mimic (miR-320c) and treatment with oxaliplatin. Each sample was stained to check for nuclear (DAPI; blue) and unrepaired DNA damage (gamma-H2AX; red).
  • B It is a result showing the percentage of damaged cells.
  • C After treatment with 250 ⁇ M oxaliplatin or 3'DW for 1 hour, and after a repair period of 48 hours, MDA-MB-231 cells were subjected to alkaline comet analysis.
  • D The results of tail moments measured using a microscope are shown.
  • E Representative images of RAD51 foci formation in MDA-MB-231 cells are shown.
  • F The number of foci per cell is shown.
  • FIG. 6 shows the results that the miR-320c mimic down-regulates the drug response in vivo.
  • A The results of immunohistochemical staining used to measure Chk1 expression in xenograft tumors are shown. Each sample was stained to identify nuclei (DAPI; blue) and Chk1 (green).
  • B NC mimic- and 3'DW-treated, NC mimic- and oxaliplatin-treated, miR-320c mimic- and 3'DW-treated, miR-320c mimic- and oxaliplatin-treated xenograft mouse tumors The ICC intensity scores obtained from Chk1 staining are shown.
  • C The results of administration of MDA-MB-231/A cells subcutaneously to a nude mouse, followed by administration of a negative control mimic (NC) and miR-320c mimic to the tumor are shown.
  • D The tumor size of each group is shown.
  • E After sacrifice of the mouse, the measured tumor weight is shown.
  • F Before the mice are sacrificed, the measured weight of each group is shown.
  • GH Immunohistochemical staining results for ⁇ -H2AX measurement and percentage histograms of ⁇ -H2AX positive cells are shown. Each sample was stained to identify the nuclei (DAPI; blue) and ⁇ -H2AX (red). ⁇ -H2AX; gamma-H2A histone member X.
  • I A schematic diagram of CHEK1 regulated by miR-320c in TNBC is shown.
  • the present inventors confirmed that miR-320c improves the reactivity of TNBC to oxaliplatin by regulating CHEK1 expression and DDR, and completed the present invention.
  • the present invention provides a biomarker composition for predicting reactivity to platinum-based anticancer drugs in triple negative breast cancer patients comprising miR-320c as an active ingredient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • the present invention provides a composition for predicting the reactivity of a platinum-based anticancer agent in patients with triple negative breast cancer, comprising an agent capable of measuring the expression level of miR-320c as an active ingredient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • the present invention provides a kit for predicting reactivity to platinum-based anticancer drugs in patients with triple negative breast cancer comprising the composition.
  • primer refers to a nucleic acid sequence having a short free 3'hydroxyl group, capable of forming a base pair with a complementary template, and acting as a starting point for template strand copying. Refers to the nucleic acid sequence. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. PCR conditions, the length of the sense and antisense primers can be appropriately selected according to techniques known in the art.
  • the term "probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to a few hundred bases capable of specifically binding in addition to mRNA, and is labeled so that the presence or absence of a specific mRNA, expression You can check the amount.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA (DNA) probe, a double strand DNA (DNA) probe, an RNA probe, or the like. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art.
  • methods for measuring the expression level of miR-320c include RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA; RNase protection assay), Northern blotting, DNA chip, Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony Immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS or protein chip may be used, but the present invention is not limited thereto.
  • sample isolated from triple negative breast cancer patients refers to tissues, cells, whole blood that differ from the control group in the expression level of the miR-320c, a biomarker for predicting the reactivity of platinum-based anticancer drugs in patients with triple negative breast cancer. , Serum, plasma, saliva, sputum, cerebrospinal fluid, or a sample such as urine, but is not limited thereto.
  • predicting reactivity to platinum-based anticancer drugs means predicting whether a patient will respond favorably or unfavorably to platinum-based anticancer drugs, or predicting the risk of resistance to platinum-based anticancer drugs. It means predicting the patient's prognosis, that is, recurrence, metastasis, survival, or disease-free survival after treatment with platinum-based anticancer drugs.
  • the present invention provides a pharmaceutical composition for enhancing the anticancer effect of a platinum-based anticancer agent in a triple negative breast cancer patient comprising a miR-320c, miR-320c expression promoter or an activator as an active ingredient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • the miR-320c expression promoter or activator may be a compound, peptide, aptamer, or antibody that specifically binds to miR-320c, but is not limited thereto.
  • the composition inhibits the expression of Checkpoint kinase 1 (CHEK1) and increases apoptosis of triple negative breast cancer cells, thereby increasing sensitivity to platinum-based anticancer agents.
  • CHEK1 Checkpoint kinase 1
  • the term "peptide” has an advantage of high binding power to a target substance, and denaturation does not occur even during thermal/chemical treatment.
  • the molecular size is small, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a high molecular protein chain, it can be used as a diagnostic kit and a drug delivery material.
  • aptamer refers to a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity.
  • DNA DNA, RNA or modified nucleic acid
  • aptamers are composed of polynucleotides that are more stable than proteins, have simple structures, and are easy to synthesize, while being able to specifically bind to antigenic substances in the same way as antibodies. I can.
  • the term "antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site.
  • the antibody in the present invention refers to an antibody that specifically binds to miR-320c of the present invention, and an antibody can be prepared according to a conventional method in the art.
  • the form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included.
  • the antibody refers to a complete form with two full-length light chains and two full-length heavy chains.
  • the antibody includes special antibodies such as humanized antibodies.
  • the kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate to which a label to develop color is conjugated by reaction with a substrate, a color developing substrate solution to react with the label, a washing solution, and It may contain an enzyme reaction stop solution, etc., and may be prepared as a plurality of separate packaging or compartments including the reagent components used.
  • the pharmaceutical composition or complex formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may be an excipient, a disintegrant, a sweetening agent, a binder, a coating agent, an expanding agent, and a lubricant.
  • a solubilizing agent such as a lubricant or a flavoring agent may be used.
  • the pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition, including at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.
  • Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • the pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of active compounds.
  • the pharmaceutical composition of the present invention is in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered as.
  • the effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of a disease.
  • the present invention provides a health functional food composition for enhancing anticancer effect against platinum-based anticancer agents in triple negative breast cancer patients comprising a miR-320c, miR-320c expression promoter or activator as an active ingredient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • the health functional food composition of the present invention may further include one or more additives selected from the group consisting of organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar and sorbitol.
  • the organic acid is not limited thereto, but may be citric acid, humic acid, adipic acid, lactic acid or malic acid
  • the phosphate is not limited thereto, but may be sodium phosphate, potassium phosphate, acid pyrophosphate or polyphosphate (polyphosphate)
  • the agent is not limited thereto, but may be a natural antioxidant such as polyphenol, catechin, alpha-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid or phytic acid.
  • the health food is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and natural flavoring agents, flavoring agents, coloring agents, and thickening agents (cheese, chocolate, etc.) in addition to the active ingredients.
  • the food composition according to an embodiment of the present invention may contain pulp for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage.
  • the formulation of the health food is not limited thereto, but may be in the form of solid, powder, granules, tablets, capsules, liquids, or beverages.
  • the present invention miR-320c, miR-320c expression promoter or activator; And it provides a pharmaceutical composition for preventing or treating triple negative breast cancer comprising a platinum-based anticancer agent as an active ingredient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating triple negative breast cancer in patients with increased expression of miR-320c comprising a platinum-based anticancer agent as an active ingredient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • the present invention comprises the steps of (1) contacting the test substance to triple negative breast cancer cells; (2) measuring the expression level of miR-320c in triple negative breast cancer cells contacted with the test substance; And (3) selecting a test substance having an increased expression level of miR-320c compared to a control sample.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • test substance used while referring to the screening method of the present invention refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. do.
  • the sample includes, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.
  • the present invention comprises the steps of selecting a patient with triple negative breast cancer with an increased expression level of miR-320c; And administering a platinum-based anticancer agent to the selected triple negative breast cancer patient.
  • the platinum-based anticancer agent may be oxaliplatin, cisplatin, or carboplatin, but is not limited thereto.
  • HEK293T, SK-BR-3, MCF7, MDA-MB-468, HS578T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, WelGENE, South Korea), and MDA-MB-231, BT-20, HCC-1937, ZR -75-1, BT-549, T47D cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (RPMI, WelGENE). 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (WelGENE) were added to the culture medium.
  • FBS fetal bovine serum
  • WelGENE penicillin/streptomycin
  • MCF-10A cells were cultured in a growth medium consisting of DMEM:F12 (1:1) in 5% horse serum (Invitrogen), 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin (cholera toxin) and 10 ug/mL insulin (Sigma-Aldrich) were added. All cell lines were cultured at 37° C., 5% CO 2 under atmospheric conditions. In addition, all cell lines were used for DNA fingerprinting analysis (Korean Cell Line Bank) in 2018.
  • miRVana TM miR-320c mimic (Ambion, USA) was reverse-transfected at a final concentration of 30 nM using siPORT TM NeoFX TM transfection agent (Ambion, USA) according to the manufacturer's instructions.
  • miRVana TM miR-negative control mimic (Negative control #1, Ambion, USA) was transfected.
  • a miRNA lentiviral vector was used to stably overexpress miR-320c in HS578T and MDA-MB-231 cells.
  • the miR-320c overexpression construct and related negative control construct were purchased from Abm® (Richmond).
  • HEK293T cells were transfected with the construct and lentiviral packaging vector (Abm®) by lentifectin (Abm®) according to the manufacturer's protocol, and viruses were collected 48 hours after transfection.
  • HS578T and MDA-MB-231 cells were infected with miR-320c overexpressing virus or negative control virus, and selected through puromycin (Sigma) and GFP.
  • the miR-320c expression level of the infected cells was confirmed through miRNA expression analysis.
  • the produced stabilized cell line was cultured in DMEM (WelGENE) to which 10% fetal bovine serum (FBS, Gibco) and puromycin (1 ⁇ g/ml) (Sigma) were added.
  • RNAs were isolated using Trizol® (Ambion, USA) according to the manufacturer's instructions.
  • Quantitative RT-PCR for miRNA expression analysis was performed with Taqman® Universal PCR Master Mix (Applied Biosystems) using LightCycler® System (Roche), and RNU48 was used as an internal control.
  • PCR conditions are as follows. The pre-reaction process (95° C., 10 minutes) and the amplification process (95° C., 10 minutes and 60° C., 30 seconds) are 45 times.
  • MRNAs for quantitative RT-PCR were extracted according to the manufacturer's instructions using NucleoSpin® RNA/Protein kit (MACHEREY-NAGEL). Using M-MLV Reverse Transcriptase (Promega, USA), 10pM oligo-dT, 2.5nM dNTP mixture and RNAse inhibitor, 5ug mRNAs were reverse transcribed. 100ng cDNA was used as a template, and qPCR was performed using SYBR Green qPCR Master Mix (PCR Biosystem, London, UK) and LightCycler® System (Roche). Each target gene was detected using specific primers. The relative expression level of the target gene or miRNA was calculated according to the 2 ⁇ (- ⁇ Cq) method and normalized to the level of 18s rRNA or RNU48 used as an intrinsic control.
  • Protein was purified using NucleoSpin® RNA/Protein kit (MACHEREY-NAGEL) and RIPA buffer. Protein content was measured using Bicinchoninic acid Solution (Sigma) and Copper (II) sulfate solution (Sigma). Proteins were separated on 15% or 8% SDS-PAGE gels. Western blotting was performed in a standard way.
  • the primary antibodies used in the present invention were Chk1 (Santa Cruz Biotechnology, USA) and ⁇ -actin (Bethyl laboratories, USA). ⁇ -actin was used as a loading control. The immunoreactive protein was detected with a horseradish peroxidase-conjugated secondary antibody and an improved chemiluminescent reagent, EzWestLumi plus (ATTO, JAPAN).
  • Human CHEK1 3'UTR containing the expected miR-320c seed sequence was amplified from MDA-MB-231 cDNA via PCR.
  • the 3'UTR of each gene containing the mutant seed sequence was amplified.
  • the amplified PCR fragments were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA) using In-fusion® HD Cloning Kit (Clontech Laboratories, USA).
  • Using Lipofectamine 2000 reagents (Invitrogen) the luciferase constructs were co-transfected into HEK293T cells with the miR-320c mimic or miR-NC mimic.
  • the Dual-Luciferase Reporter Assay System Promega, according to the manufacturer's instructions, luciferase activity was measured after 48 hours.
  • Alkaline Comet Assay Alkaline Comet Assay
  • DNA damage was induced through oxaliplatin treatment.
  • a solid oxaliplatin stock was purchased from Sigma Aldrich , dissolved in H 2 O to prepare 10 mM. DNA damage was detected according to the manufacturer's protocol (4250-050-K, Trevigen) through the Alkaline comet assay kit. Briefly, 1 ⁇ 10 5 /ml of trypsinized cells and LMAgarose dissolved at 37°C were mixed at a ratio of 1:10 (v/v). Then, the mixture was embedded on CometSlides at 4° C. for 10 minutes. The slides were immersed in Lysis Solution overnight at 4°C. The next day, the slides were immersed in alkaline unwinding solution for 1 hour at 4°C in the dark. Then, electrophoresis was performed for 30 minutes. Next, the slides were washed and stained with SYBR for 30 minutes. Images were obtained with a confocal microscope (Nikon). Analysis was commissioned by KORMED.
  • colony formation analysis was performed by the following method. 0.5-1 x 10 3 /ml cells were seeded on 6-well plates. Thereafter, oxaliplatin (Sigma) was treated for each concentration. In the control plate, until the cells formed colonies of appropriate size (at least 50 cells per colony), the cells were incubated for 1-3 weeks at 37°C in a CO 2 incubator. After incubation, the cells were fixed and stained with 0.5% crystal violet solution (including 10% ethanol).
  • the prepared cells were trypsinized and washed with PBS. Thereafter, cells were suspended in 100 ⁇ l of 1 ⁇ Binding Buffer of FITC Annexin V Apoptosis Detection Kit I (BD PharmingenTM), and stained with FITC and Pi. Then, the cells were analyzed by flow cytometry (FACS Canto II, BD). A total of 30,000 cells were counted for each sample. The stabilizing cell line overexpressing miR-320c was tagged with GFP, and Sub-G1 analysis was performed. The prepared cells were trypsinized and washed with PBS. Then, the cells were suspended in 500 ⁇ l PBS and fixed overnight with cold 70% ethanol.
  • mice were injected with synthetic miRNA into the tumor, and oxaliplatin (Tocris Bioscience) or 3′DW was administered intraperitoneal (ip).
  • oxaliplatin Tocris Bioscience
  • 3′DW intraperitoneal
  • Several forms of synthetic miRNA miR-320c/miR-NC) (6.25 ⁇ g) mixed with 1.6 ⁇ L siPORTTM NeoFXTM Transfection Agent (Ambion) dissolved in 50 ⁇ L PBS were injected into the tumor every 3-5 days for each tumor.
  • 3'DW or oxaliplatin was administered ip (2mg/kg) at intervals of 3-5 days.
  • the tumor size was measured each time using a caliper at intervals of 3 to 5 days, and the tumor volume was calculated by the following equation. Length ⁇ width 2 ⁇ 0.5. Mice were sacrificed with excess anesthetic and tumors were harvested for immunohistochemical analysis and other analyses.
  • the present inventors performed an experiment according to the method shown in FIG. 1.
  • the present inventors used TNBC patient tissue microRNA microarray data and identified a group of miRNAs that are down-regulated in TNBC tissues compared to adjacent normal tissues (FIG. 1B).
  • FIG. 1B adjacent normal tissues
  • the present inventors confirmed that the expression of miRNA-320c was significantly reduced in TNBC.
  • the present inventors confirmed the expression of miR-320c in breast cancer cell lines, it was observed that miR-320c expression was decreased in TNBC compared to normal breast cancer cell line MCF10A as well as non-TNBC breast cancer cell line (FIG. 1C).
  • GEO dataset analysis was performed.
  • the expression level of miR-320c was decreased in the TNBC subtype compared to other non-TNBC subtypes (Fig. 1D).
  • Kaplan-Meier analysis was performed. Only in the TNBC patient group, low hsa-miR-320c expression was observed, which was significantly associated with a low overall survival rate (Fig. 1E).
  • Kaplan-Meier analysis using TCGA pan-cancer atlas data also identified five cancer types, including breast cancer, showing a significant correlation between low miR-320c expression and low survival rate. Taken together, these results indicate that miR-320c is downregulated in TNBC, which may be a potential marker in cancer.
  • mRNA microarray data was inserted and analyzed.
  • TargetScan http://www.targetscan.org
  • 847 candidate genes with the conserved miR-320c-binding sequence were discovered.
  • the present inventors selected 7 candidate genes, which showed a significant negative correlation with miR-320c expression, and was predicted through miRDB to have a miR-320c target sequence in 3'UTR (Fig. 2A).
  • the TCGA dataset (Cancer Genome Atlas, 2012) was used, and CHEK1 expression was significantly increased in TNBC (FIG. 2B). Additionally, CHEK1 was upregulated in TNBC compared to other breast cancer subtypes in the METABRIC dataset (Fig. 2C).
  • CHEK1 was included in the 122 gene candidate groups, which were commonly predicted as targets of miR-320c in all four databases, and it was found that the miR-320c-binding sequence was present.
  • TargetScan Agarwal et al, 2015
  • miRwalk 2.0 Dweep & Gretz, 2015
  • DIANA-microT web server v5.0 Paraskevopoulou et al, 2013; Reczko et al, 2012
  • miRDB Liu & Wang, 2019; Wong & Wang, 2015
  • the mRNA expression level of CHEK1 showed a tendency to increase overall in the TNBC cell line (Fig. 2D). Additionally, the Chk1 protein level also showed a tendency to increase in the TNBC cell line (Fig. 2E).
  • CHEK1 mRNA expression analysis was performed. Breast cancer subtypes were classified by pam50. As a result, it was confirmed that the CHEK1 mRNA level in the four types of cancer with low survival rate was similar to that in the breast, showing low miR-320c expression.
  • the present inventors confirmed whether CHEK1 is regulated by miR-320c overexpression in the TNBC cell line.
  • the present inventors performed a dual luciferase analysis using a CHEK1 3'UTR construct containing a miR-320c binding seed sequence region.
  • the target recognition ability of the microRNA is dependent on the seed sequence in the 3'UTR region of the target.
  • Each of the miR-320c sequence and the complementary seed sequence regions of miR-320c in CHEK1 3'UTR obtained using TargetScan are shown in FIG. 2F.
  • the luciferase activity of the wild-type CHEK1 3'UTR construct was greatly attenuated by miR-320c overexpression. However, none of the mutant-type CHEK1 3'UTR constructs inhibited luciferase activity (Fig. 2G).
  • the present inventors used three TNBC cell lines (MDA-MB-231, MDA-MB-468 and HS578T) transfected with miR-320c mimic. When miR-320c was overexpressed (FIG. 2H), the expression of CHEK1 mRNA and protein was suppressed in all three TNBC cell lines as expected (FIG. 2I and FIG. 2J). Taken together, CHEK1 in TNBC tissues and TNBC cell lines showed a significant negative correlation with miR-320c mRNA and protein, indicating that it is regulated by miR-320c.
  • the present inventors tried to confirm whether miR-320c regulates the DNA damage response by regulating CHEK1.
  • Chk1 was known to act on the phosphorylation of various effective factors involved in checkpoint, DNA repair and apoptosis. Meanwhile, Chk1 was found to inhibit caspase-2 apoptosis response to DNA damage.
  • the present inventors used oxaliplatin, a platinum-based compound.
  • the present inventors confirmed that CHEK1-upregulated, miR-320c-downregulated MDA-MB-231 cells exhibited lower apoptosis compared to MCF7 breast cancer cells exhibiting opposite expression patterns.
  • the present inventors performed FACS analysis by staining with FITC and PI.
  • MDA-MB-231 cells showed a lower apoptosis cell population than MCF7 (FIGS. 3A and 3B ).
  • the present inventors confirmed whether ectopic miR-320c can induce apoptosis by regulating the expression of CHEK1 by FACS analysis together with FITC and PI staining.
  • miR-320c mimic transfection miR-320c was successfully increased (Fig. 3C), but when oxaliplatin treatment and miR-320c mimic transfection were performed, the number of apoptotic cell populations in the TNBC cell line increased most.
  • DNA damage induces homologous recombinant repair (HRR) by phosphorylation of RAD51 through Chk1.
  • HRR homologous recombinant repair
  • the present inventors tried to confirm the role of miR-320c by regulating CHEK1 expression in the repair of oxaliplatin-induced DNA damage.
  • the present inventors treated MDA-MB-231 TNBC cells with a high concentration of oxaliplatin for 1 hour, followed by a repair period of 48 hours.
  • the present inventors performed immunocytochemistry (ICC) staining with ⁇ -H2AX (H2AX phosphorylation), a DNA double-helix cleavage marker.
  • the present inventors performed an alkaline comet analysis to detect DNA cleavage induced by DNA damage in MDA-MB-231 and HS578T TNBC cells.
  • Cells were transfected with miR-320c mimic or negative control (NC) mimic.
  • NC negative control
  • DNA damage was increased in miR-320c mimic-transfected cells compared to NC mimic-transfected cells (FIGS. 4C and 4D).
  • CHEK1 was knocked down with CHEK1 siRNA.
  • the present inventors performed ICC staining with RAD51 antibody in order to detect RAD51 collected at the DNA damage site.
  • Treatment with oxaliplatin increased RAD51 foci formation.
  • miR-320c mimic transfection in oxaliplatin-treated TNBC cells induced a decrease in RAD51 foci (FIGS. 4E and 4F ).
  • reduction in RAD51 foci formation was also confirmed in CHEK1-siRNA transfected TNBC cells.
  • the present inventors tried to confirm whether miR-320c can affect the reactivity to oxaliplatin by regulating CHEK1 expression. Accordingly, a colony formation assay was performed to measure oxaliplatin reactivity in vitro. First, the effectiveness of oxaliplatin was compared using two different types of breast cancer cells showing different expression patterns of CHEK1 and miR-320c. Consistent with the previous results, MCF7 cells were more effective against oxaliplatin compared to MDA-MB-231 cells (FIGS. 5A and 5B ).
  • the present inventors measured the Chk1 level using immunofluorescence (IF). Chk1 levels were significantly down-regulated by administration of the miR-320c mimic (FIGS. 6A and 6B ), and the mean tumor volume of the miR-320c mimic treated group was lower compared to the NC mimic treated group. In addition, the tumor volume of the group treated with both miR-320c mimic and oxaliplatin was the lowest compared to other groups (FIGS. 6C and 6D ). Additionally, this trend was also observed in tumor weight (Fig. 6E). However, as a result of weight comparison between each group, there was no significant difference (FIG. 6F).
  • IF immunofluorescence
  • gamma H2AX levels were significantly increased in the tumor group treated with both the miR-320c mimic and oxaliplatin (FIGS. 6G and 6H ). Taking the above results together, the upregulation of miR-320c by administration of mimics improved the effect of oxaliplatin treatment.
  • the present inventors have discovered the role of miR-320c in a novel regulatory mechanism of CHEK1 in TNBC (Fig. 6I).
  • CHEK1 When oxaliplatin was treated with TNBC cells, CHEK1 was successfully activated through DNA damage induction. Activated Chk1 inhibited DNA damage-induced apoptosis and was involved in RAD51 recruitment to DNA damage sites for DNA repair.
  • miR-320c was overexpressed by the miR-320c mimic, Chk1 could not be sufficiently translated.
  • apoptosis was increased and RAD51 recruitment decreased in TNBC cells upregulated miR-320c compared to TNBC cells with less miR-320c expression. This weakened DNA repair and accumulated DNA damage.
  • miR-320c upregulation made TNBC cells more sensitive to oxiliplatin.

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Abstract

La présente invention concerne une composition de biomarqueur comprenant miR-320c en tant que principe actif pour la prédiction de la réactivité à un médicament d'un patient atteint d'un cancer du sein triple négatif à un agent anticancéreux à base de platine. Les présents inventeurs ont découvert que la régulation directe de l'expression de CHEK1 par miR-320c, qui est régulée à la baisse dans la plupart des lignées cellulaires TNBC. À travers de nouvelles recherches, les présents inventeurs ont révélé que la restauration ectopique de miR-320c dans des cellules TNBC traitées par oxaliplatine supprime une réponse liée à l'endommagement d'ADN et à la réparation d'ADN, améliore l'instabilité génomique, et active l'apoptose par la régulation négative de CHEK1. En outre, une combinaison d'un mimétique de miR-320c et d'oxaliplatine inhibe efficacement la progression tumorale. Les résultats montrent que miR-320c joue un rôle important dans la réactivité des dellules TNBC à l'oxaliplatine par régulation de l'expression de CHEK1, ce qui implique que, en tant que cible thérapeutique pour augmenter la réactivité à la chimiothérapie, miR-320c est attendu pour trouver des applications avantageuses dans une stratégie thérapeutique pour le cancer du sein triple négatif.
PCT/KR2020/007605 2019-10-18 2020-06-11 Composition de biomarqueur comprenant mir-320c en tant que principe actif pour la prédiction de la réactivité à un médicament d'un patient atteint d'un cancer du sein triple négatif à un agent anticancéreux à base de platine Ceased WO2021075662A1 (fr)

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Citations (4)

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EP2354246A1 (fr) * 2010-02-05 2011-08-10 febit holding GmbH ARNm dans le diagnostic du cancer ovarien
KR20140104419A (ko) * 2011-11-25 2014-08-28 인테그라겐 Egfr 억제제를 사용한 치료에 대한 반응을 예측하는 방법
WO2015171510A2 (fr) * 2014-05-05 2015-11-12 The Regents Of The University Of California Microarn (miarn) circulants utilisables en tant que biomarqueurs de la rétinopathie diabétique (rd) et de la dégénérescence maculaire liée à l'âge (dmla)
WO2019115748A1 (fr) * 2017-12-14 2019-06-20 Unicyte Ev Ag Vehicules pharmaceutiques contenant des arnmi pour leur utilisation dans le traitement du cancer du rein

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EP2354246A1 (fr) * 2010-02-05 2011-08-10 febit holding GmbH ARNm dans le diagnostic du cancer ovarien
KR20140104419A (ko) * 2011-11-25 2014-08-28 인테그라겐 Egfr 억제제를 사용한 치료에 대한 반응을 예측하는 방법
WO2015171510A2 (fr) * 2014-05-05 2015-11-12 The Regents Of The University Of California Microarn (miarn) circulants utilisables en tant que biomarqueurs de la rétinopathie diabétique (rd) et de la dégénérescence maculaire liée à l'âge (dmla)
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