KR102645036B1 - Composition for diagnosing anti-cancer medicine resistance and kit comprising the same - Google Patents

Abstract

The present invention relates to a composition and kit for diagnosing anticancer drug resistance. The composition, kit, and method of the present invention can effectively predict treatment responsiveness to an anticancer drug and enable selective and individualized anticancer drug treatment for cancer patients.

Description

Composition for diagnosing anti-cancer medicine resistance and kit comprising the same}

The present invention relates to a composition for diagnosing anticancer drug resistance and a kit containing the same.

Chemotherapy is an expensive treatment that generally causes toxic side effects in the human body and is economically expensive. In particular, because significant improvement in survival rate due to actual anticancer drug treatment is observed only in a very small number of patients, recent efforts are underway to select patient groups that respond to anticancer drugs from the perspective of individualized therapy.

For example, Mammaprint or OncotypeDx, commercialized kits using prognostic factors for breast cancer patients, are representative results of research to select patients with poor prognosis who require chemotherapy among patients with early breast cancer.

Recently, discoveries have been made to identify substances that change immediately after the use of anticancer drugs (acquired resistance related markers) or substances related to anticancer drug response/resistance inherent in the tumor (intrinsic resistance related markers) that can confirm the effectiveness of preoperative chemotherapy at an earlier stage. Various attempts are being made for this purpose, and most of these studies are being conducted on a preoperative chemotherapy platform.

Accordingly, in the treatment of cancer, there is a need to develop new targets that can be used to predict treatment responsiveness of anticancer drugs, and although research on this is being conducted, it is still at a minimal level.

Korean Patent Publication No. 10-2017-0109787

The purpose of the present invention is to provide a composition and kit for diagnosing anticancer drug resistance.

The purpose of the present invention is to provide a method for providing information for diagnosing anticancer drug resistance.

The purpose of the present invention is to provide a screening method for candidate anticancer drug resistance inhibitors.

The purpose of the present invention is to provide a pharmaceutical composition for suppressing anticancer drug resistance.

1. A composition for diagnosing anticancer drug resistance containing a substance that specifically binds to NMI (N-Myc interator) mRNA or its protein.

2. The composition of 1 above, wherein the substance is at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

3. The composition of 1 above, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.

4. A kit for diagnosing anticancer drug resistance containing the composition of any one of items 1 to 3 above.

5. A method of providing information for the diagnosis of anticancer drug resistance, including the step of measuring the expression level of NMI (N-Myc interator) mRNA or its protein in a sample isolated from a diagnostic subject.

6. The method of 5 above, wherein the diagnosis subject is a breast cancer patient.

7. The method of 5 above, wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, or urine.

8. The method of 5 above, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.

9. A screening method for an anticancer drug resistance inhibitor candidate comprising the step of selecting a substance that reduces the expression of NMI (N-Myc interator) mRNA or its protein in a sample isolated from a diagnostic subject.

10. The method of item 9 above, wherein the diagnosis subject is a breast cancer patient.

11. The method of item 9 above, wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, or urine.

12. The method of item 9 above, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.

13. A pharmaceutical composition for suppressing anticancer drug resistance containing a substance that reduces the expression of NMI (N-Myc interator) mRNA or its protein.

14. In item 13 above, the substances include low molecular weight drugs, gene drugs, protein drugs, extracts, nucleic acids, oligonucleotides, proteins, peptides, antibodies, RNA, DNA, PNA, aptamers, chemicals, enzymes, amino acids, and sugars. , a composition that is at least one selected from the group consisting of lipids.

15. The composition of item 13 above, wherein the substance is a Nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor.

16. The composition of item 13 above, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.

The present invention relates to a composition and kit for diagnosing anticancer drug resistance. The composition, kit, and method of the present invention can effectively predict treatment responsiveness to an anticancer drug and enable selective and individualized anticancer drug treatment for cancer patients.

Figure 1A is a diagram confirming the expression of NMI by IHC score in the complete response and non-complete response patient groups, and Figure 1B is the result of immunohistochemical staining of cancer tissue of a breast cancer patient with anticancer drug resistance. The brown area indicates NMI expression.
Figure 2 is a diagram confirming drug responsiveness according to inhibition of NMI expression.
Figure 3 is a diagram showing tamoxifen responsiveness according to inhibition of NMI expression.
Figure 4 is a diagram confirming the ability to form spheroids due to inhibition of NMI expression.
Figure 5 is a diagram confirming the efficacy of inhibiting NMI expression by ML385 (NRF2 inhibitor).

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for diagnosing anticancer drug resistance containing a substance that specifically binds to NMI (N-myc interactor) mRNA or its protein.

NMI is present in a sample derived from a diagnostic target, and its mRNA or protein sequence can be a known sequence in NCBI genbank, etc. For example, in the case of humans, NMI mRNA may have the sequence of SEQ ID NO: 1, and the protein may have the sequence of SEQ ID NO: 2. The sequence is not limited to this, and the sequence of the individual species to be diagnosed can be used.

The diagnostic subjects include animals that currently have cancer or have had cancer, animals that are suspected of having cancer, and other animals for which information for anticancer drug resistance diagnosis is requested. The animals include mammals, including humans. It can be.

The above cancers include breast cancer, liver cancer, lung cancer, bladder cancer, stomach cancer, uterine cancer, colon cancer, colon cancer, blood cancer, ovarian cancer, prostate cancer, pancreas cancer, spleen cancer, testicular cancer, thymus cancer, brain cancer, esophagus cancer, kidney cancer, biliary tract cancer, It may be at least one selected from ovarian cancer, kidney cancer, thyroid cancer, or skin cancer, and preferably breast cancer, but is not limited thereto.

The sample is separated from the diagnostic object, and may be, for example, tissue, cells, blood, serum, plasma, saliva, or urine. Specifically, it may be cancer tissue separated from the diagnostic object, but is not limited thereto.

The substance may be a substance that detects the NMI mRNA or its protein in a sample isolated from a diagnostic subject.

The substance is not limited as long as it can detect NMI mRNA or its protein, but may be, for example, one selected from the group consisting of antibodies, aptamers, DNA, proteins, and polypeptides.

As used herein, “antibody” refers to a protein molecule specific for an antigenic site. For the purposes of the present application, an antibody refers to an antibody that specifically binds to NMI, the marker protein, and may include all monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.

The monoclonal antibody may be produced using a hybridoma method or phage antibody library technology well known in the art, but may not be limited thereto.

The polyclonal antibody can be produced by a method well known in the art, which includes injecting the protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. These polyclonal antibodies can be produced from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc., but may not be limited thereto.

In addition, the antibodies herein may also include special antibodies such as chimeric antibodies, humanized antibodies, and human antibodies.

The “peptide” has the advantage of high binding capacity to the target substance and does not undergo denaturation even during heat/chemical treatment. Additionally, because the molecule size is small, it can be used as a fusion protein by attaching it to another protein. Specifically, it can be used by attaching it to a polymer protein chain, so it can be used as a diagnostic kit and drug delivery material.

The "aptamer" refers to a special type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. It refers to a type of polynucleotide. As mentioned above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easier to synthesize, so they can be used as a replacement for antibodies. You can.

Substances that specifically bind to the mRNA may be sense and antisense primers, or probes, but are not limited thereto.

In the present invention, 'primer' refers to a short gene sequence that serves as the starting point for DNA synthesis and an oligonucleotide synthesized for use in diagnosis, DNA sequencing, etc. The primers can generally be synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and can be modified by methylation, capping, etc. by known methods.

In the present invention, “probe” refers to a nucleic acid that can specifically bind to mRNA of a few bases to several hundred bases in length, produced through enzyme-chemical separation purification or synthesis. The presence or absence of mRNA can be confirmed by labeling it with a radioactive isotope or enzyme, and it can be designed, modified and used by known methods.

Since the nucleotide sequence of the gene encoding the NMI is known, a probe or primer that specifically binds to the nucleotide sequence of the gene encoding the NMI, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide sequence, those skilled in the art will use the sequence as the basis. The primer or probe can be designed according to conventional methods in the art.

The anticancer drugs can be used without limitation as long as they are effective in treating cancer, and include alkylating agents, antimetabolites, antitumor antibiotics, anthracyclines, and plant alkaloids. , topoisomerase inhibitors, monoclonal antibodies, anti-tumor hormones, and mitotic spindle inhibitors, specifically doxetaxel and paclitaxel. , doxorubicin, cyclophosphamide, and tamoxifen.

The composition of the present invention is a substance used to predict before administration whether anticancer drug administration may be useful in the treatment of cancer in a target patient, and is treated with a sample isolated from a diagnostic target to determine the expression level of NMI mRNA or protein in the sample. can be used to predict reactivity to anticancer drugs by measuring .

For example, by comparing the expression level of NMI mRNA or its protein in the sample of a patient suspected of anticancer drug resistance with the expression level of a patient in complete response after anticancer drug administration, the NMI expression level in the suspected patient is compared to the NMI expression level in the complete response patient. If it is high, it can be judged to be resistant to anticancer drugs compared to patients in complete remission.

The present invention provides a kit for diagnosing anticancer drug resistance comprising the composition.

The kit not only contains a material that specifically binds to NMI mRNA or its protein, but also includes one or more other components, compositions, solutions, or devices suitable for the analytical method for measuring the expression level of NMI mRNA or its protein used in the kit. It can be included.

If the kit is for measuring the expression level of NMI mRNA or protein, it may be a kit containing the essential elements necessary to perform RT-PCR. In addition to each pair of primers specific for the mRNA of the marker gene, the RT-PCR kit also contains test tubes or other suitable containers, reaction buffer, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, and RNase. It may include inhibitors, DEPC-water (dEPC water), sterilized water, etc. Additionally, it may include a pair of primers specific to the gene used as a quantitative control.

The kit includes a substrate, a suitable buffer, and a suitable buffer for the immunological detection of a substance that specifically binds to the nucleotide sequence of the gene encoding NMI, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence. It may include a solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate. 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, and the coloring enzyme may be peroxidase, alkaline phosphatase ( alkaline phosphatase) can be used, the fluorescent substance can be FITC, RITC, etc., and the chromogenic substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), etc. may be used.

The kit may be a microarray for diagnosing anticancer drug resistance that can measure the expression level of NMI mRNA. The microarray can be easily prepared by a person skilled in the art according to methods known in the art using the indicator factors, and according to one embodiment, the mRNA of the gene encoding the SIRT1, SIRT3 or SIRT6 protein or a fragment thereof It may be a microarray in which cDNA of the corresponding sequence is attached to a substrate as a probe.

In addition, the kit of the present application includes an antibody that specifically binds to the marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with the substrate, a color-generating substrate solution to react color with the label, a washing solution, and an enzyme. It may contain a reaction stopping solution, etc., and may be manufactured into a number of separate packaging or compartments containing the reagent components used, but may not be limited thereto.

The kit herein may include not only an agent capable of measuring the expression level of NMI mRNA or its protein in a patient sample, but also one or more types of compositions, solutions, or devices suitable for expression level analysis. For example, the kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of an antibody, but may not be limited thereto.

As a specific example, the kit may be a kit characterized by including essential elements required to perform ELISA in order to implement various ELISA methods such as ELISA kit and sandwich ELISA. These ELISA kits contain specific antibodies against the above proteins. The antibody is an antibody with high specificity and affinity for NMI and little cross-reactivity to other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. Additionally, ELISA kits may include antibodies specific for control proteins. Other ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances capable of binding to antibodies. It may not be limited to this.

In addition, the kit may be a kit for implementing Western blot, immunoprecipitation analysis, complement fixation analysis, flow cytometry, or protein chip, and may further include additional components suitable for each analysis method. Through these analysis methods, anticancer drug resistance can be diagnosed by comparing the amount of antigen-antibody complex formation.

Provides an information provision method for diagnosing anticancer drug resistance, including the step of measuring the expression level of NMI (N-Myc interator) mRNA or its protein in a sample isolated from a diagnostic subject.

The measurement may be performed by treating the sample with a substance that detects NMI mRNA or its protein.

The substance that detects the NMI mRNA or its protein may be within the range exemplified above.

The sample, diagnostic object, and anticancer agent are as described above.

As a method of measuring the expression level of the NMI mRNA or its protein, a method of measuring the concentration in the sample of mRNA, which is a transcript of the gene encoding NMI, or the concentration of the NMI protein in the sample may be selected, but is not limited to this, It can be performed by selecting a method commonly used in the technical field of the present invention.

Methods for measuring the concentration of the mRNA in the sample include reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), Examples include, but are not limited to, RNase protection assay (RPA), Northern blotting, and DNA chip.

As a method of measuring the concentration of the protein in a sample, the amount of the protein can be confirmed using an antibody that specifically binds to the protein. Analysis methods for this include immunorubbing test, sandwich assay, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunodiffusion method. , rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), Western blotting, fluid cytometry ( flow cytometry, enzyme substrate color development method, antigen-antibody agglutination method, and protein chip, etc. Preferably, ELISA (enzyme linked immunosorbent assay) can be used, but is not limited thereto.

The information provision method of the present invention may further include comparing the expression level with that of a control subject.

The information provision method of the present invention compares the expression level of NMI mRNA or its protein in the sample of a patient suspected of anticancer drug resistance with the expression level of a patient in complete response after anticancer drug administration according to the above method, and the NMI expression level in the suspected patient shows complete response. If it is higher than the patient's NMI expression level, it can provide information that can be used to determine that the patient has anticancer drug resistance compared to patients in complete remission. In addition, if the expression level of the NMI mRNA or its protein is measured to be similar or lower than that of a patient in complete remission, information can be provided to determine that there is no anticancer drug resistance.

Provided is a screening method for a candidate anticancer drug resistance inhibitor, including the step of selecting a substance that reduces the expression of NMI (N-Myc interator) mRNA or its protein in a sample isolated from a diagnostic subject.

In the above method, anticancer drug resistance is suppressed if the expression level of the mRNA or protein is reduced by treating the test material to the subject, and such test material can be selected as a candidate for an anticancer drug resistance inhibitor.

In addition, the method can be used to treat samples isolated from individuals with anticancer drug resistance with a test substance and compare the expression level of NMI mRNA or protein before and after treatment, or to treat some of several individuals with the test substance to compare the comparison group and the above. Expression levels can also be compared.

The test substance may be a newly synthesized or known substance and may include without limitation substances expected to be effective in suppressing anticancer drug resistance, and to test whether it affects the expression level of the mRNA or protein of the present invention. Refers to an unknown substance used in screening. The test substances include, but are not limited to, chemicals, nucleotides, antisense-RNA, shRNA (short hairpin RNA), siRNA (small interference RNA), and natural product extracts.

In one embodiment of the present invention, the substance that reduces the expression level of NMI mRNA or its protein may be siRNA or shRNA. For example, when the diagnostic subject is a human, siNMI of SEQ ID NO: 3 and 4 can be used, shNMI of SEQ ID NO: 5, or shNMI of SEQ ID NO: 6 can be used. The sequence is not limited to this, and siNMI or shNMI of the individual species being diagnosed can be used.

In addition, diagnostic objects, samples, and anticancer agents are the same as described above.

The present invention provides a pharmaceutical composition for suppressing anticancer drug resistance containing a substance that reduces the expression of NMI (N-Myc interator) mRNA or its protein.

Substances that reduce the expression level of the NMI mRNA or its protein include low molecular weight drugs, gene drugs, protein drugs, extracts, nucleic acids, oligonucleotides, proteins, peptides, antibodies, RNA, DNA, PNA, aptamers, chemicals, and enzymes. It may be at least one selected from the group consisting of amino acids, sugars, lipids, compounds (natural compounds and/or synthetic compounds), and elements constituting them, but the expression of the mRNA of the gene encoding NMI or the protein encoded by the gene There is no particular limitation as long as it is a substance that has a reducing effect.

The substance may be a nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor. It may be an antisense nucleotide, siRNA, or shRNA that binds complementary to the mRNA of the NRF2 gene, or it may be a compound, peptide, peptide mimetics, aptamer, antibody, or natural product that specifically binds to the NRF2 protein. , but is not limited to this.

For example, the NRF2 inhibitor may be ML385 (CAS No 846557-71-9). It can be seen that when NMI overexpressing cell line is treated with ML385 according to an embodiment of the present invention, NMI expression is suppressed.

The pharmaceutical composition of the present invention can be administered in combination with an anticancer agent. In this case, the effect of suppressing anticancer drug resistance and the treatment effect of the anticancer drug can be obtained at the same time, thereby maximizing the effect of the anticancer chemotherapy.

The composition may further include a pharmaceutically acceptable carrier and may be formulated together with the carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The composition of the present invention can be applied in any formulation containing a substance that reduces the expression of NMI mRNA or its protein as an active ingredient, and can be prepared as an oral or parenteral formulation. The pharmaceutical formulation of the present invention may be administered orally, rectally, nasally, topically (including cheeks and under the tongue), subcutaneously, vaginally, or parenterally (intramuscularly, subcutaneously). and forms suitable for administration (including intravenous) or forms suitable for administration by inhalation or insufflation.

The composition of the present invention is administered in a pharmaceutically effective amount. The effective dosage level depends on factors including the type and severity of the patient's disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. can be decided. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The dosage of the composition of the present invention varies widely depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The appropriate dosage is, for example, the patient's It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy of the substance used to reduce the expression of the NMI mRNA or its protein. In general, it can be calculated based on the EC50 measured to be effective in in vivo animal models and in vitro, for example, it may be 0.01 μg to 1 g per kg of body weight, and in unit periods of daily, weekly, monthly or yearly, It can be administered once or several times per unit period, or it can be administered continuously for a long period of time using an infusion pump. The number of repeated doses is determined by considering the time the drug stays in the body and the concentration of the drug in the body. Even after treatment, depending on the course of disease treatment, the composition may be administered for recurrence.

The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar functions in relation to the treatment of anticancer drug resistance, or a compound that maintains/increases the solubility and/or absorption of the active ingredient. Additionally, optionally, it may further include a chemotherapy agent, an anti-inflammatory agent, an antiviral agent, and/or an immunomodulatory agent.

Additionally, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Dosage forms may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

Hereinafter, the present invention will be described in detail with reference to examples.

experiment material

1. Cell culture

Breast cancer cell lines MCF7, MCF7/PR (MCF7-paclitaxel resistant cell line), MCF7/ADR (MCF7-adriamycin resistant cell line), MCF7/TAMR (MCF7-tamoxifen resistant cell line), MDA-MB-231, BT20, MDA-MB-468 All cell lines were cultured in DMEM medium + 10% FBS (fetal bovine serum, Gibco) + 1% PS (Penicillin streptomycin, Gibco) in an incubator at 37°C (5% CO ₂ ).

2. NMI gene suppression using siRNA (siRNA transfection)

For NMI gene knock-down, siNMI (SEQ ID NO: 3 and 4) and siControl (Bioneer, Korea) were purchased, and each siRNA was used in various breast cancer cell lines (MCF7, MCF7/MCF7) using RNAimax (thermofisher) according to the manufacturer's instructions. PR, MCF7/ADR, MCF7/TAMR, MDA-MB-231, BT20, MDA-MB-468) were transfected, and the inhibition of NMI gene expression was confirmed by RT-PCR and western blot.

3. Establishment of NMI-inhibiting stable cell line

To establish a stable cell line overexpressing NMI, pLOC (Precision LentiORF control vector, lentiviral control vector (Dhrmacon)) and NMI vector (lentivirus-based vector, sequencing primer used for NMI amplification Forward 5'CTCCATAGAAGACACCGAC-3' (SEQ ID NO. 7) , Reverse 5-'CATATAGACAAACGCACAC3' (SEQ ID NO: 8))), To establish an NMI-silencing stable cell line, shcontrol (GIPZ non-silencing shRNA control) and shNMI (SEQ ID NO: 5 or 6) were purchased from Dharmacon and added to the HEK293T cell line, respectively. lentiviral transfection was performed using the vector, and 24 and 48 hours after transfection, each cell culture was collected and transduced into the target cell line, followed by Blasticidin (overexpression cell line - Hs578T) or puromycin selection (suppressor cell line - BT20, NMI overexpression, inhibition and control stable cell lines were established through MDA-MB-231).

Experiment method and results

1. Confirmation of differences in NMI expression in breast cancer patients responding to anticancer drugs and non-responding patients

Among breast cancer patients who received neoadjuvant chemotherapy, there were a chemotherapy complete response group (n=54, meaning a group in which tumor cells completely disappeared, CR) and a chemotherapy non-remission group (n=56, nCR). In order to confirm the difference in NMI expression, the expression score of NMI was scored based on H-score (Ishibashi H et.al, 2003) (Table 1). Neoadjuvant chemotherapy was performed on patients who received Docetaxel, Adriamycin, and Cyclophosphamide. Immunohistochemical staining was performed using the NMI antibody from Novus (cat. NBP1-90374).

The results are shown in Figure 1, and the IHC score between the complete response (CR) group and the non-remission (nCR) group was confirmed to be 0.855:1.275 ( p =0.012).

2. Confirmation of drug response according to inhibition of NMI expression

Cell lines suppressing NMI expression (MCF7/PR, MDA-MB-231, BT20, MDA-MB-468) were seeded at 3,000 cells/well in a 96-well plate, and 24 hours later, each drug (docetaxel = 20nM, paclitaxel = 20nM, Adriamycin = 500nM, cyclophosphamide = 4mM), and cell viability (%) was measured 48 hours after drug treatment. Cell viability was measured using the cellTiter-Glo luminescent cell viability assay (Promega) kit, and luminescence was measured using the GloMax luminometer (Promega).

As a result, it was confirmed that drug responsiveness to four anticancer drugs (DTX, PTX, ADR, CPM) increased in NMI expression suppressed cell lines (Figure 2).

3. Confirmation of tamoxifen response according to NMI expression

Responsiveness to tamoxifen was measured using ER (estrogen receptor) positive cell lines (MCF7, MCF7/PR, MCF7/ADR). ER-positive cell lines were seeded at 3,000 cells/well in a 96-well plate and treated with tamoxifen (12.5 μM, 25 μM) 24 hours later, and cell survival rate was measured 48 hours after tamoxifen treatment.

Referring to Figure 3A, when doxorubicin and paclitaxel resistance developed in the MCF7 cell line, NMI expression increased. Referring to Figure 3B, it can be seen that responsiveness to tamoxifen increases when NMI is inhibited.

4. Evaluation of 3D spheroid formation ability according to NMI expression (3D spheroids formation assay)

In general, when resistance to a drug is acquired, 3D spheroids formation ability is promoted. To confirm the difference in the ability to form 3D spheroids when NMI expression is suppressed, the expression of NMI was suppressed using siRNA, and 1,000 cells/cell were incubated in a low-attached 96-well plate to form 3D spheroids of siControl and siNMI cells. Well cells were seeded and cultured for 7-10 days in spheroid assay medium containing bFGF (20nM), EGF (20nM), and 1XB27 supplements. Afterwards, the number of three-dimensional spheroids with a size of 100㎛ or more was counted and tabulated.

Referring to Figure 4, since spheroid formation was significantly suppressed in the cell line in which the expression of NMI was suppressed, it can be seen that suppressing NMI expression increases drug responsiveness. Additionally, this suggests that NMI is associated with drug resistance.

5. Confirmation of NMI inhibition efficacy by ML385

In order to confirm the correlation between NRF2 and NMI, the relationship between the two proteins was confirmed on the bc-GenExMiner v4.3 ( http://bcgenex.centregauducheau.fr/BC-GEM/GEM-Accueil.php?js=1 ) site. (Figure 5A). In a total of n = 9414 data samples, NMI and NRF2 were positively correlated (r = 0.30, p < 0.0001). To confirm this, it was confirmed by RT-PCR in NMI expression suppressed stable cell lines (BT20, MDA-MB-231) and NMI overexpressed stable cell lines (Hs578T) (Figure 5B).

In addition, NRF2-specific inhibitor (ML385) was treated with BT20-shControl cells and stable cell lines suppressing shNMI expression at 25 μM and 50 μM for 24 hours, and NMI expression was confirmed through western blot method. As a result, it can be seen that NMI expression is suppressed by ML385, an NRF2 inhibitor (Figure 5C).

<110> SEOUL NATIONAL UNIVERSITY HOSPITAL <120> Composition for diagnosing anti-cancer medicine resistance and kit comprising the same <130> 19P10048 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1229 <212> RNA <213> Homo sapiens <400> 1 gtttcaggcg ctgctgtttt ccgggaaggg caggcgcgct gggccttggg gagctgcgct 60 cggcgggcgg acgcggggga tcatggaagc tgataaagat gacacacaac aaattcttaa 120 ggagcattcg ccagatgaat ttataaaaga tgaacaaaat aagggactaa ttgatgaaat 180 tacaaagaaa aatattcaac taaagaagga gatccaaaag cttgaaacgg agttacaaga 240 ggctaccaaa gaattccaga ttaaagagga tattcctgaa acaaagatga aattcttatc 300 agttgaaact cctgagaatg acagccagtt gtcaaatatc tcctgttcgt ttcaagtgag 360 ctcgaaagtt ccttatgaga tacaaaaagg acaagcactt atcacctttg aaaaagaaga 420 agttgctcaa aatgtggtaa gcatgagtaa acatcatgta cagataaaag atgtaaatct 480 ggaggttacg gccaagccag ttccattaaa ttcaggagtc agattccagg tttatgtaga 540 agtttctaaa atgaaaatca atgttactga aattcctgac acattgcgtg aagatcaaat 600 gagagacaaa ctagagctga gcttttcaaa gtcccgaaat ggaggcggag aggtggaccg 660 cgtggactat gacagacagt ccgggagtgc agtcatcacg tttgtggaga ttggagtggc 720 tgacaagatt ttgaaaaaga aagaataccc tctttatata aatcaaacct gccatagagt 780 tactgtttct ccatacacag aaatacactt gaaaaagtat cagatatttt caggaacatc 840 taagaggaca gtgcttctga caggaatgga aggcattcaa atggatgaag aaattgtgga 900 ggatttaatt aacattcact ttcaacgggc aaagaatgga ggtggagaag tagatgtggt 960 caagtgttct ctaggtcaac ctcacatagc atactttgaa gaatagactt aacagaatca 1020 tgaaaactat agctttttaa cccggattac tgtaaatgtt tgacaaaaat gaatatgctt 1080 ttccttaaaa aatgaaaact ttaattttta ccatccattt atgtttagat acaaaactta 1140 tttccatgtt tctgaatctt ctttgtttca aatggtgctg catgttttca actacaataa 1200 gtgcactgta ataaaaagtt ttgtttata 1229 <210> 2 <211> 307 <212> PRT <213> Homo sapiens <400> 2 Met Glu Ala Asp Lys Asp Asp Thr Gln Gln Ile Leu Lys Glu His Ser 1 5 10 15 Pro Asp Glu Phe Ile Lys Asp Glu Gln Asn Lys Gly Leu Ile Asp Glu 20 25 30 Ile Thr Lys Lys Asn Ile Gln Leu Lys Lys Glu Ile Gln Lys Leu Glu 35 40 45 Thr Glu Leu Gln Glu Ala Thr Lys Glu Phe Gln Ile Lys Glu Asp Ile 50 55 60 Pro Glu Thr Lys Met Lys Phe Leu Ser Val Glu Thr Pro Glu Asn Asp 65 70 75 80 Ser Gln Leu Ser Asn Ile Ser Cys Ser Phe Gln Val Ser Ser Lys Val 85 90 95 Pro Tyr Glu Ile Gln Lys Gly Gln Ala Leu Ile Thr Phe Glu Lys Glu 100 105 110 Glu Val Ala Gln Asn Val Val Ser Met Ser Lys His His Val Gln Ile 115 120 125 Lys Asp Val Asn Leu Glu Val Thr Ala Lys Pro Val Pro Leu Asn Ser 130 135 140 Gly Val Arg Phe Gln Val Tyr Val Glu Val Ser Lys Met Lys Ile Asn 145 150 155 160 Val Thr Glu Ile Pro Asp Thr Leu Arg Glu Asp Gln Met Arg Asp Lys 165 170 175 Leu Glu Leu Ser Phe Ser Lys Ser Arg Asn Gly Gly Gly Glu Val Asp 180 185 190 Arg Val Asp Tyr Asp Arg Gln Ser Gly Ser Ala Val Ile Thr Phe Val 195 200 205 Glu Ile Gly Val Ala Asp Lys Ile Leu Lys Lys Lys Glu Tyr Pro Leu 210 215 220 Tyr Ile Asn Gln Thr Cys His Arg Val Thr Val Ser Pro Tyr Thr Glu 225 230 235 240 Ile His Leu Lys Lys Tyr Gln Ile Phe Ser Gly Thr Ser Lys Arg Thr 245 250 255 Val Leu Leu Thr Gly Met Glu Gly Ile Gln Met Asp Glu Glu Ile Val 260 265 270 Glu Asp Leu Ile Asn Ile His Phe Gln Arg Ala Lys Asn Gly Gly Gly 275 280 285 Glu Val Asp Val Val Lys Cys Ser Leu Gly Gln Pro His Ile Ala Tyr 290 295 300 Phe Glu Glu 305 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Homo sapiens siNMI <400> 3 cuccauacac agaaauaca 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Homo sapiens siNMI <400> 4 catatagaca aacgcacac 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Homo sapiens shNMI 1 <400> 5 tcttcatcca tttgaatgc 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Homo sapiens shNMI 2 <400> 6 ttaatggaac tggcttggc 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NMI Primer_F <400> 7 ctccatagaa gacaccgac 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NMI Primer_R <400> 8 catatagaca aacgcacac 19

Claims (16)

A composition for diagnosing anticancer drug resistance containing a substance that specifically binds to NMI (N-Myc interator) mRNA or its protein.
The composition according to claim 1, wherein the substance is at least one selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.
The composition of claim 1, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.
A kit for diagnosing anticancer drug resistance comprising the composition of any one of claims 1 to 3.
A method of providing information for diagnosing anticancer drug resistance, comprising measuring the expression level of NMI (N-Myc interator) mRNA or its protein in a sample isolated from a diagnostic subject.
The method of claim 5, wherein the diagnosis subject is a breast cancer patient.
The method of claim 5, wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, or urine.
The method of claim 5, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.
A screening method for a candidate anticancer drug resistance inhibitor, comprising the step of selecting a substance that reduces the expression of NMI (N-Myc interator) mRNA or its protein in a sample isolated from a diagnostic subject.
The method of claim 9, wherein the diagnosis subject is a breast cancer patient.
The method of claim 9, wherein the sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, or urine.
The method of claim 9, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.
A pharmaceutical composition for suppressing anticancer drug resistance containing a substance that reduces the expression of NMI (N-Myc interator) mRNA or its protein.
The method of claim 13, wherein the substances include low molecular weight drugs, genetic drugs, protein drugs, extracts, nucleic acids, oligonucleotides, proteins, peptides, antibodies, RNA, DNA, PNA, aptamers, chemicals, enzymes, amino acids, sugars, and lipids. A composition, which is at least one selected from the group consisting of.
The composition of claim 13, wherein the substance is a Nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor.
The composition of claim 13, wherein the anticancer agent is selected from the group consisting of doxetaxel, paclitaxel, doxorubicin, cyclophosphamide, and tamoxifen.