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WO2024053887A1 - Marqueur génique copine1 pour le pronostic des métastases du cancer du sein her2-positif et son utilisation - Google Patents

Marqueur génique copine1 pour le pronostic des métastases du cancer du sein her2-positif et son utilisation Download PDF

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WO2024053887A1
WO2024053887A1 PCT/KR2023/012001 KR2023012001W WO2024053887A1 WO 2024053887 A1 WO2024053887 A1 WO 2024053887A1 KR 2023012001 W KR2023012001 W KR 2023012001W WO 2024053887 A1 WO2024053887 A1 WO 2024053887A1
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breast cancer
her2
copine1
positive breast
gene
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English (en)
Korean (ko)
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유재철
최혜영
우동균
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경상국립대학교산학협력단
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Publication of WO2024053887A1 publication Critical patent/WO2024053887A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to the Copine1 gene marker for predicting metastatic prognosis of HER2-positive breast cancer and its use.
  • Breast cancer is the cancer with the highest incidence and mortality rate in women. In the case of early breast cancer, the survival rate reaches 90%, but in the case of late-stage breast cancer, the survival rate drops sharply. Therefore, early detection when there are no symptoms can improve the survival rate. It's a method.
  • the most basic treatment for breast cancer is surgical resection of the lesion, and after surgery, auxiliary treatments such as chemotherapy, radiation therapy, and anti-hormone therapy are performed.
  • auxiliary treatments such as chemotherapy, radiation therapy, and anti-hormone therapy are performed.
  • the most important factors that determine the prognosis of breast cancer are the size of the cancer mass, whether it has metastasized to lymph nodes, or whether it has metastasized throughout the body, and in the case of stage 4 breast cancer that has metastasized, the 5-year survival rate is reported to be less than 20%.
  • Cancer metastasis is the proliferation of cancer cells in a place other than the tissue or organ in which they originally originated. To achieve this, cancer cells must penetrate the surrounding tissue and move to nearby blood vessels, or form new blood vessels and move through them to other organs. , This is called invasion.
  • Breast cancer is broadly classified into three types depending on the expression of estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). If ER or PR is expressed in breast cancer tissue, it is classified as hormone receptor positive breast cancer. If ER or PR is not expressed but HER2 is expressed, it is classified as HER2 positive breast cancer. If ER, PR, and HER2 are not expressed, it is classified as triple negative breast cancer. Hormone receptor positive breast cancer is further classified into luminal A type or luminal B type. Symptoms and treatments are different for each type of breast cancer, and it is known that among all breast cancers, hormone receptor-positive breast cancer accounts for 60%, HER2-positive breast cancer accounts for 25%, and triple-negative breast cancer accounts for 15%.
  • ER estrogen receptor
  • PR progesterone receptor
  • HER2 human epidermal growth factor receptor 2
  • Hormone receptor-positive breast cancer is known to grow slowly and be less aggressive, while HER2-positive breast cancer and triple-negative breast cancer are known to grow rapidly and be more aggressive.
  • HER2-positive breast cancer has a poor prognosis due to frequent recurrence and metastasis, research on HER2 protein-targeted treatments and prediction of metastasis prognosis for HER2-positive breast cancer is steadily underway.
  • Korean Patent Publication No. 2022-0010871 discloses 'Marker for diagnosing or predicting prognosis of metastatic breast cancer and its use' using the FBXL16 gene
  • Korean Patent No. 1888193 discloses 'Circle periostin as a diagnostic indicator of breast cancer metastasis'.
  • the ' Copine1 gene marker for predicting metastasis prognosis of HER2 positive breast cancer and its use' of the present invention.
  • the present invention was derived from the above needs, and the present inventors overexpressed the Copine1 gene in HER2-positive breast cancer cells (SK-BR3) and luminal A type breast cancer cells (MCF-7) and then performed cell invasion analysis. .
  • HER2-positive breast cancer cells SK-BR3
  • the number of infiltrated cells when the Copine1 gene was overexpressed was confirmed to significantly increase compared to when the Copine1 gene was not overexpressed, whereas the number of infiltrated cells was significantly increased compared to when the Copine1 gene was not overexpressed.
  • case 7 it was confirmed that there was no significant difference in the number of infiltrated cells when the Copine1 gene was overexpressed and when the Copine1 gene was not overexpressed.
  • the present invention was completed by confirming the possibility of the Copine1 gene as a biomarker for predicting the prognosis of HER2-positive breast cancer-specific metastasis.
  • the present invention provides a biomarker composition for predicting metastasis prognosis of HER2-positive breast cancer containing the Copine1 gene as an active ingredient.
  • the present invention provides a composition for predicting the prognosis of metastasis of HER2-positive breast cancer, including an agent for measuring the expression level of the mRNA or protein thereof of the Copine1 gene.
  • the present invention provides a kit for predicting metastasis prognosis of HER2-positive breast cancer, comprising the composition.
  • the present invention provides the following steps: (1) measuring the expression level of the mRNA of the Copine1 gene or the protein expressed from the gene from an isolated biological sample of a HER2-positive breast cancer patient; (2) comparing the expression level with a control sample; and (3) determining that the risk of metastasis of HER2-positive breast cancer is high when the expression level is higher than that of the control sample.
  • the present invention provides a method for treating HER2-positive breast cancer, comprising measuring the mRNA expression level of the Copine1 gene or the expression level of the protein encoded by the gene after administration of a candidate substance expected to inhibit metastasis of HER2-positive breast cancer.
  • a method for screening metastasis inhibitors is provided.
  • the present invention provides a pharmaceutical composition for inhibiting metastasis of HER2-positive breast cancer, comprising an inhibitor of Copine1 protein expression as an active ingredient.
  • the Copine1 gene or its protein of the present invention is a biomarker for predicting the metastatic prognosis of HER2-positive breast cancer, and is expected to be useful in predicting the metastatic prognosis of HER2-positive breast cancer by analyzing the expression level of the Copine1 gene or its protein. .
  • Figure 1 shows the results of immunohistochemical staining for Copine1 protein using tissue from a HER2-positive breast cancer patient and a tissue from a luminal A type breast cancer patient (A), and the mRNA expression of the Copine1 gene using mRNA expression profile data from breast cancer patients. This is the result of comparing expression levels (B).
  • Figure 2 shows the results of confirming the expression of Copine1 protein after overexpressing the Copine1 gene in HER2-positive breast cancer cells (SK-BR3) and luminal A type breast cancer cells (MCF-7) (A) and the results of cell invasion analysis. It is (B, C).
  • GFP Control group
  • CPNE1 Copine1 gene overexpression vector insertion group
  • scale bar 100 ⁇ m.
  • Figure 3 shows the results of confirming the expression of Copine1 protein (A) and cell invasion analysis after inhibiting the expression of the Copine1 gene in HER2-positive breast cancer cells (SK-BR3) and luminal A type breast cancer cells (MCF-7). This is one result (B, C).
  • GFP control group
  • CPNE1 vector insertion group for overexpression of the Copine1 gene
  • shCPNE1 vector insertion group for inhibition of Copine1 gene expression
  • scale bar 100 ⁇ m.
  • the present invention provides a biomarker composition for predicting metastasis prognosis of HER2-positive breast cancer containing the Copine1 gene as an active ingredient.
  • the present invention provides a composition for predicting the prognosis of metastasis of HER2-positive breast cancer, including an agent for measuring the expression level of the mRNA or protein thereof of the Copine1 gene.
  • the Copine1 gene of the present invention may preferably consist of the base sequence of SEQ ID NO: 1, but is not limited thereto. Additionally, homologs of the above base sequence are included within the scope of the present invention. Specifically, the gene includes a base sequence having sequence homology of at least 70%, preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence of SEQ ID NO: 1. can do.
  • the “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .
  • Copine1 protein may consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
  • the term 'metastasis' refers to a state in which a tumor is transplanted from its primary site to another part of the body along various routes and settles and proliferates there. Since the metastasis of cancer is not only determined by the unique characteristics of the cancer, but is also the most important clue in determining the prognosis of cancer, it is treated as the most important clinical information related to the survival of cancer patients.
  • prognosis refers to an increase or decrease in the severity of HER2 positive breast cancer.
  • prognosis usually refers to the presence or absence of metastasis or survival period within a certain period of time after the onset of cancer or surgical procedure. Prediction of prognosis provides clues about the direction of future treatment, including whether or not to receive chemotherapy, especially for HER2-positive breast cancer patients. It is a method that predicts the course of treatment after comprehensively considering the patient's physiological or environmental status after treatment of the disease. It can be interpreted as an action.
  • the 'metastasis prognosis prediction' means predicting the risk of metastasis in HER2-positive breast cancer patients by predicting in advance whether HER2-positive breast cancer will metastasize. For example, predicting a ‘good prognosis’ means that HER2-positive breast cancer patients have a low risk of metastasis, meaning that HER2-positive breast cancer patients are more likely to be cured, while predicting a ‘poor prognosis’ means that HER2-positive breast cancer patients have a low risk of metastasis.
  • the risk of metastasis in breast cancer patients is high, meaning that HER2-positive breast cancer patients are more likely to have their cancer metastasize, recur, or die.
  • 'mRNA expression level measurement' is a process of confirming the presence and expression level of mRNA of the HER2-positive breast cancer metastasis marker gene ( Copine1 ) in a biological sample to predict the metastasis prognosis of HER2-positive breast cancer.
  • This can be known by measuring the amount of mRNA. Analysis methods for this include reverse transcriptase polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT-PCR), real time quantitative RT-PCR (RT-PCR), and RNase protection assay (RPA). ), northern blotting, DNA chip, etc., but are not limited to these.
  • the agent for measuring the mRNA expression level of the Copine1 gene is not limited thereto, but is preferably a primer, probe, or antisense oligonucleotide, and the primer, probe, or antisense oligonucleotide is a base sequence of the Copine1 gene ( With reference to GenBank no.BC001142), a person skilled in the art can design it using known methods, programs or tools.
  • the term 'primer' refers to a nucleic acid sequence with a short free 3' hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. refers to a short nucleic acid sequence that functions as a Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.
  • nucleoside triphosphates and reagents for polymerization i.e., DNA polymerase or reverse transcriptase
  • the prognosis of metastasis of HER2-positive breast cancer can be predicted by performing PCR amplification using sense and antisense primers that can bind to the base sequence of the Copine1 gene and determining whether the desired product is produced.
  • PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.
  • the term 'probe' refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundred bases, capable of forming a specific binding to mRNA, and is labeled to confirm the presence or absence of a specific mRNA.
  • Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc.
  • hybridization is performed using a probe complementary to the Copine1 polynucleotide, and the prognosis of metastasis of HER2-positive breast cancer can be predicted based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
  • 'protein expression level measurement' is a process of confirming the presence and expression level of a protein expressed in the HER2-positive breast cancer metastasis marker gene ( Copine1 ) in a biological sample to predict the metastasis prognosis of HER2-positive breast cancer. This can be typically performed by checking the amount of protein. Analysis methods for this include ELISA (enzyme linked immunosorbent assay), western blot, radioimmunoassay (RIA), radioimmunodiffusion, ouchterlony immunodiffusion, and rocket. ) include, but are not limited to, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, FACS (fluorescence activated cell sorting), and protein chip.
  • the agent for measuring the protein expression level is not limited thereto, but may preferably be an antibody or aptamer that specifically binds to the Copine1 protein.
  • the term 'antibody' is a term known in the art and refers to a specific protein molecule directed to an antigenic site.
  • an antibody refers to an antibody that specifically binds to the Copine1 protein, which is a marker of the present invention, and such Copine1 antibody is obtained by cloning the entire length or part of the Copine1 gene into an expression vector according to a conventional method. It can be produced by a conventional method by obtaining a protein encoded by the full length or part of the Copine1 gene and using the obtained protein as an immunogen (antigen).
  • the form of the antibody of the present invention is not particularly limited, and polyclonal antibodies, monoclonal antibodies, or functional fragments having antigen binding properties are also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the functional fragment of the antibody molecule refers to a fragment that possesses at least an antigen-binding function and includes Fab, F(ab'), F(ab')2, and ScFv.
  • the term 'aptamer' refers to a nucleic acid that can specifically and strongly bind to a specific molecule while maintaining a stable tertiary structure. Because of its specific binding function, it is compared to antibodies and is evaluated as an alternative technology to antibodies.
  • the present invention also provides a kit for predicting metastasis prognosis of HER2-positive breast cancer, including the composition for predicting metastasis prognosis of HER2-positive breast cancer.
  • the kit of the present invention can predict the metastasis prognosis of HER2-positive breast cancer by checking whether the mRNA of the Copine1 gene or the protein expressed therefrom is detected.
  • the kit for predicting the metastatic prognosis of HER2-positive breast cancer of the present invention includes an agent for detecting the expression level of the Copine1 gene (e.g., a primer or probe) or an agent that can specifically detect a protein (e.g., an antibody or aptamer). )
  • an agent for detecting the expression level of the Copine1 gene e.g., a primer or probe
  • an agent that can specifically detect a protein e.g., an antibody or aptamer.
  • one or more other component compositions, solutions, or devices suitable for the analysis method may be included.
  • the kit for measuring the mRNA expression level of the Copine1 gene may be a kit containing the essential elements required to perform RT-PCR.
  • the RT-PCR kit consists of test tubes or other suitable containers, reaction buffer (of varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, in addition to each primer pair specific for the marker gene. It may contain the same enzyme, DNase, RNase inhibitor, DEPC-water, sterilized water, etc. Additionally, it may include a pair of primers specific to the gene used as a quantitative control.
  • kits of the present invention may be a prediction kit containing essential elements required to perform DNA chip processing.
  • a DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, agents, enzymes, etc. for producing a fluorescent label probe. Additionally, the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof.
  • the kit of the present invention when the kit of the present invention is a kit for measuring the expression level of Copine1 protein, it includes a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody. can do.
  • the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass
  • the coloring enzyme may be peroxidase or alkaline phosphatase.
  • Fatase alkaline phosphatase
  • fluorescein isothiocyanate FITC
  • rhodamine B isothiocyanate RITC
  • ABTS 2,2'-azino-bis (3-ethyl) is used as a coloring substrate solution.
  • Benzothiazoline-6-sulfonic acid or OPD (o-phenylenediamine) or TMB (tetramethylbenzidine) can be used.
  • the present invention also,
  • control group may be a breast cancer sample in which metastasis has not progressed, but is not limited thereto.
  • the expression level of the mRNA or protein of the Copine1 gene from the biological sample can be measured by isolating the mRNA or protein of the gene from the biological sample, and the method of isolating the mRNA or protein of the gene is a method known in the art. It can be performed using .
  • the term 'biological sample' includes, but is not limited to, samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, or urine, and the biological sample may be used in a manipulated or unmanipulated state. there is.
  • the expression level of the mRNA of the Copine1 gene or the protein expressed from the gene measured from the separated biological sample of the HER2-positive breast cancer patient is compared to the control group. If the expression level of the mRNA of the Copine1 gene or the protein expressed from the gene measured in the sample is higher, the HER2-positive breast cancer patient can be judged to have a high risk of metastasis.
  • the present invention also provides a method for treating metastasis of HER2-positive breast cancer, comprising measuring the expression level of the Copine1 gene or the expression level of the protein encoded by the gene after administration of a candidate substance expected to inhibit metastasis of HER2-positive breast cancer.
  • a method for screening inhibitors is provided.
  • a HER2-positive breast cancer metastasis inhibitor can be screened by comparing the change (increase or decrease) in the expression level of the Copine1 gene or Copine1 protein in the presence and absence of the HER2-positive breast cancer metastasis inhibitor candidate, preferably Copine1 Substances that indirectly or directly reduce the expression level of the gene or Copine1 protein can be selected as metastasis inhibitors for HER2-positive breast cancer. That is, the expression level of the Copine1 gene or Copine1 protein of the present invention is measured in biological samples from HER2-positive breast cancer patients in the absence of the HER2-positive breast cancer metastasis inhibitor candidate, and the Copine1 protein of the present invention is measured in the presence of the HER2-positive breast cancer metastasis inhibitor candidate.
  • the expression level of the Copine1 gene or Copine1 protein of the present invention in the presence of the HER2-positive breast cancer metastasis inhibitor candidate is compared to the expression level in the absence of the HER2-positive breast cancer metastasis inhibitor candidate. Substances that reduce this level can be predicted as metastasis inhibitors for HER2-positive breast cancer.
  • the present invention also provides a pharmaceutical composition for inhibiting metastasis of HER2-positive breast cancer comprising an inhibitor of Copine1 protein expression as an active ingredient.
  • the Copine1 protein expression inhibitor is an antisense nucleotide, dsRNA, small interfering RNA (siRNA), or short hairpin that binds complementary to the mRNA of the Copine1 gene. It may be RNA (short hairpin RNA, shRNA), a compound that specifically binds to the Copine1 protein, a peptide, a peptide mimetics, an aptamer, an antibody, or a natural product, but is not limited thereto.
  • tissue from a breast cancer patient was donated by the Korea Tumor Tissue Bank (Hwasun, Korea).
  • Immunohistochemical staining for Copine1 (CPNE1) protein was performed using 11 tissue samples from patients with HER2-positive breast cancer and 11 tissue samples from patients with luminal A type breast cancer. Each breast cancer tissue was placed in 0.3% H 2 O 2 and reacted for 30 minutes, then placed in blocking buffer (5% FBS, 0.3% Triton X-100) and incubated with anti-CPNE1 antibody (1:100, Santa Cruz Biotechnology , USA) was used to react overnight at 4°C.
  • the mRNA expression profile data of breast cancer patients were downloaded from the TCGA database and used.
  • the mRNA expression profile data of a total of 526 breast cancer tissues and 61 normal tissues (non-tumor tissues) were aligned using the Agilent 244K custom gene expression platform (G4502A-07-3; Agilent, USA), then quantile normalized and t- Through the assay, the mRNA expression level of the Copine1 gene in total breast cancer tissue and normal tissue was compared.
  • Copine1 ( CPNE1 ) gene
  • the cDNA sequence of the Copine1 gene (GenBank no.BC001142) was inserted into the pDEST-Ad-GFP (pDEST-adenovirus-green fluorescent protein) vector using the Gateway Cloning System (Thermo Fisher, USA) to clone Copine1.
  • a vector for gene overexpression (pDEST-Ad-GFP-CPNE1) was prepared.
  • a specific shRNA (5'-CGCTTTGGAATCTATGACATA-3': SEQ ID NO: 3) sequence targeting the Copine1 gene was inserted in front of the pDEST-Ad-GFP vector skeleton to create a vector for inhibiting Copine1 gene expression (pDEST-Ad-shCPNE1). was manufactured.
  • Human breast cancer cell lines SK-BR3 and MCF-7 (Table 1) were purchased from the Korea Cell Line Bank and cultured in DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. ) were cultured in medium at 37°C and 5% CO 2 and used for experiments.
  • DMEM Dulbecco Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • Cells used in the present invention cell line molecular characteristics subtype SK-BR3 ER-, PR-, HER2+ HER2 positive MCF-7 ER+, PR+, HER2- Luminal A
  • ER estrogen receptor
  • PR progesterone receptor
  • HER2 human epidermal growth factor receptor 2.
  • Adenovirus was propagated in 293A cells (Korean Cell Line Bank, Korea) using the ViraPowerTM Adenoviral Expression System (Thermo Fisher Scientific), and pDEST-Ad-GFP vector, pDEST-Ad-GFP-CPNE1 vector, or pDEST-Ad- The shCPNE1 vector was transfected into 293A cells, and 10 days later, adenovirus particles were recovered.
  • Breast cancer cells were inoculated into a 6-well plate at 1 ⁇ 10 5 cells/ml, transfected with adenovirus at a multiplicity of infection (MOI) of 100, and cultured at 37°C for 48 hours.
  • MOI multiplicity of infection
  • Invasion of breast cancer cells was analyzed using a transwell chamber (8 ⁇ m pore size; BD Biosciences, USA). DMEM medium supplemented with 10% FBS was placed in the lower chamber, and 5 ⁇ 10 5 cells were suspended in DMEM medium without serum in the upper chamber. 48 hours later, the cells that moved to the lower chamber were fixed with methanol, stained with 0.2% crystal violet, and the number of infiltrated cells was measured using a light microscope (IX71; Olympus, Japan).
  • Copine1 gene was overexpressing in HER2-positive breast cancer cells (SK-BR3) and luminal A type breast cancer cells (MCF-7), the expression of Copine1 protein was confirmed, and cell invasion analysis was performed.
  • SK-BR3 HER2-positive breast cancer cells
  • MCF-7 luminal A type breast cancer cells
  • Copine1 protein was confirmed, and cell invasion analysis was performed.

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Abstract

La présente invention concerne un marqueur génique Copine1 permettant de pronostiquer les métastases d'un cancer du sein HER2-positif, ainsi que son utilisation. Plus particulièrement, la présente invention concerne une composition de biomarqueurs pour pronostiquer les métastases du cancer du sein HER2-positif, comprenant le gène Copine1 comme principe actif, une composition pour pronostiquer les métastases du cancer du sein HER2-positif, comprenant un agent pour mesurer un niveau d'expression d'ARNm ou de protéine du gène Copine1, et une composition pharmaceutique pour inhiber les métastases du cancer du sein HER2-positif, comprenant un inhibiteur de l'expression de la protéine Copine1 comme principe actif.
PCT/KR2023/012001 2022-09-05 2023-08-11 Marqueur génique copine1 pour le pronostic des métastases du cancer du sein her2-positif et son utilisation WO2024053887A1 (fr)

Applications Claiming Priority (2)

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KR1020220112252A KR20240033766A (ko) 2022-09-05 2022-09-05 HER2 양성 유방암의 전이 예후 예측용 Copine1 유전자 마커 및 이의 용도
KR10-2022-0112252 2022-09-05

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WO2024053887A1 true WO2024053887A1 (fr) 2024-03-14

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013057323A2 (fr) * 2011-10-21 2013-04-25 The Provost, Fellows, Foundation Scholars, And The Other Members Of The Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Marqueur et cible pour la sensibilité et la résistance à des agents anticancéreux
WO2015049289A2 (fr) * 2013-10-01 2015-04-09 Ait Austrian Institute Of Technology Gmbh Méthode et moyens de diagnostic du cancer du sein
KR20180103024A (ko) * 2017-03-08 2018-09-18 한양대학교 산학협력단 Her2 양성 암 및 항-her2 치료에 대한 바이오마커 및 이의 용도

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013057323A2 (fr) * 2011-10-21 2013-04-25 The Provost, Fellows, Foundation Scholars, And The Other Members Of The Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Marqueur et cible pour la sensibilité et la résistance à des agents anticancéreux
WO2015049289A2 (fr) * 2013-10-01 2015-04-09 Ait Austrian Institute Of Technology Gmbh Méthode et moyens de diagnostic du cancer du sein
KR20180103024A (ko) * 2017-03-08 2018-09-18 한양대학교 산학협력단 Her2 양성 암 및 항-her2 치료에 대한 바이오마커 및 이의 용도

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Title
CHOI HYE YOUNG, LEE HYO JUNG, MOON KYOUNG MI, MOON DONG KYU, LEE SECHAN, PARK HYEWON, HONG JINPYO, PARK MI JUNG, WOO DONG KYUN, YO: "Up-regulation of CPNE1 Appears to Enhance Cancer Progression in HER2-positive and Luminal A Breast Cancer Cells", ANTICANCER RESEARCH, INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH, GR, vol. 42, no. 7, 1 July 2022 (2022-07-01), GR , pages 3445 - 3452, XP093147402, ISSN: 0250-7005, DOI: 10.21873/anticanres.15831 *
ERIKO TOKUNAGA; YASUE KIMURA; EIJI OKI; NAOYUKI UEDA; MOTONORI FUTATSUGI; KOJIRO MASHINO; MANABU YAMAMOTO; MASAHIKO IKEBE; YOSHIHI: "Akt is frequently activated in HER2/neu‐positive breast cancers and associated with poor prognosis among hormone‐treated patients", INTERNATIONAL JOURNAL OF CANCER, JOHN WILEY & SONS, INC., US, vol. 118, no. 2, 27 July 2005 (2005-07-27), US , pages 284 - 289, XP071282666, ISSN: 0020-7136, DOI: 10.1002/ijc.21358 *
SHAO ZHIHONG, MA XIAOLONG, ZHANG YUFENG, SUN YUANYUAN, LV WENJUAN, HE KUIGANG, XIA RUI, WANG PEIJUN, GAO XIAOLONG: "CPNE1 predicts poor prognosis and promotes tumorigenesis and radioresistance via the AKT singling pathway in triple‐negative breast cancer", MOLECULAR CARCINOGENESIS, JOHN WILEY & SONS, INC., US, vol. 59, no. 5, 1 May 2020 (2020-05-01), US , pages 533 - 544, XP093147401, ISSN: 0899-1987, DOI: 10.1002/mc.23177 *
YOO, JAE-CHUL ET AL.: " Research of Copine1 with binding proteins in regulation about breast cancer proliferation and related signal pathway", FINAL (RESULT) REPORT OF INDIVIDUAL BASIC SCIENCE&ENGINEERING RESEARCH SUPPORT PROGRAM/BALANCED ACADEMIC DEVELOPMENT SUPPORT PROJECT, KR, KR, pages 1 - 7, XP009554342, Retrieved from the Internet <URL:https://scienceon.kisti.re.kr/srch/selectPORSrchReport.do?cn=TRKO202100013710> *

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