Biomarker for determining subtype of triple-negative breast cancer and use thereof

Abstract

The present invention relates to a composition for determining subtypes of triple-negative breast cancer and a composition for preventing or treating triple-negative breast cancer. By measuring the expression level of DNp63α, which is a biomarker identified in the present invention, the molecular subtypes of triple-negative breast cancer can be accurately classified. Based on the classified molecular subtypes, the therapeutic responsiveness to specific therapeutic agents is predicted with high reliability, thereby significantly contributing to early establishment of treatment strategies and improved patient survival. In addition, the composition of the present invention significantly induces apoptosis of cancer cells by inhibiting the expression of DNp63α in triple-negative breast cancer expressing DNp63α, and thus can be effectively used as an efficient therapeutic composition for triple-negative breast cancer, which is refractory solid cancer.

Description

Biomarkers for subtyping triple-negative breast cancer and their uses

[0001] The present invention relates to a method for determining a molecular subtype of triple-negative breast cancer using Delta Np63 alpha (DNp63α) protein as a biomarker.

[0002]

[0003] Triple-negative breast cancer (TNBC) is a type of breast cancer in which the expression of all three markers—estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)—is confirmed to be negative upon biopsy. It accounts for 18-25% of all breast cancers. TNCs are known to be more aggressive than other phenotypes, with frequent recurrence and metastasis, and an overall poorer prognosis. However, the precise mechanisms of onset, recurrence, and metastasis, as well as the discovery of biomarkers for diagnosis and treatment, have not been elucidated.

[0004] To date, studies have been conducted to understand triple-negative breast cancer from a molecular perspective and explore classification criteria based on phenotype. However, studies using patient-derived tissues or cell lines have limitations in accuracy due to the mixed presence of various cells in tissue samples and frequent transformations during culture.

[0005] Meanwhile, the inventors of the present invention have previously established organoids that efficiently recapitulate the phenotype of triple-negative breast cancer using surgical tissue obtained from triple-negative breast cancer patients. Using these organoids, the inventors conducted gene expression analysis and molecular cell biology experiments to characterize triple-negative breast cancer. Furthermore, they compared patient characteristics to identify reliable biomarkers and therapeutic targets for triple-negative breast cancer.

[0006] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0007]

[0008] The present inventors have devoted extensive research efforts to develop an efficient biomarker that can reliably predict the clinical characteristics of breast cancer, particularly triple-negative breast cancer (TNBC), a disease with a poor prognosis and with a poorly understood pathogenesis and treatment target. As a result, we discovered that triple-negative breast cancer can be classified into molecular subtypes with distinct pathological characteristics and phenotypes based on DNp63α protein expression, enabling the early establishment of personalized treatment strategies based on these subtypes. This discovery led to the completion of the present invention.

[0009] Therefore, the purpose of the present invention is to provide a composition for determining the subtype of triple-negative breast cancer.

[0010] Another object of the present invention is to provide a composition for preventing or treating triple-negative breast cancer.

[0011] Another object of the present invention is to provide a method for screening a composition for preventing or treating triple-negative breast cancer.

[0012] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0013] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0014]

[0015] According to one aspect of the present invention, the present invention provides a composition for determining a subtype of breast cancer, comprising as an active ingredient an agent for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding the same.

[0016] The present inventors have dedicated themselves to developing an effective biomarker that can reliably predict the clinical characteristics of breast cancer, particularly triple-negative breast cancer (TNBC), a disease with a poor prognosis and with poorly understood pathogenesis and treatment targets. As a result, we discovered that TNp63α protein expression can classify triple-negative breast cancer into molecular subtypes with distinct pathologic characteristics and phenotypes. Based on these subtypes, personalized treatment strategies can be developed early.

[0017] In the present invention, the term “subtype” or “molecular subtype” encompasses a subtype of a tissue classified according to its unique phenotype / genotype, including its susceptibility to a disease (e.g., cancer), prognosis, and therapeutic responsiveness to a specific drug, based on the gene and / or protein expression profile of the specific tissue. Tumor subtyping is very important not only for accurate diagnosis but also for predicting the biological characteristics of individual cancer tissues. Breast cancer, in particular, shows complex clinical and pathological patterns compared to other solid cancers, and among these, triple-negative breast cancer in particular has diverse biological characteristics and progression processes. Therefore, accurate molecular subtyping is a crucial issue for early establishment of patient-tailored treatment strategies.

[0018] In this specification, the term “composition for subtyping” means an integrated mixture or device including a means for measuring the expression level of DNp63α protein or a gene encoding the same for determining the subtype of breast cancer tissue, and may also be expressed as a “kit for subtyping.”

[0019] According to a specific embodiment of the present invention, the agent for measuring the expression level of the DNp63α protein is an antibody or an antigen-binding fragment thereof that specifically binds to the DNp63α protein; or an aptamer that specifically binds to the DNp63α protein.

[0020] According to the present invention, the DNp63α protein of the present invention can be detected using an immunoassay method utilizing antigen-antibody reactions and used to analyze subtypes of breast cancer tissue. This immunoassay can be performed using various immunoassay or immunostaining protocols developed in the past.

[0021] For example, when the method of the present invention is performed according to a radioimmunoassay method, antibodies labeled with radioisotopes (e.g., C14, I125, P32, and S35) may be used. In the present invention, the antibody that specifically recognizes the DNp63α protein is a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody.

[0022] The antibodies of the present invention can be produced by methods commonly practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816,567), or the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production are described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999, etc.

[0023] By analyzing the intensity of the final signal through the above-described immunoassay process, the subtype of triple-negative breast cancer can be predicted.

[0024] As used herein, the term “antigen binding fragment” means a portion of a polypeptide in the overall structure of an immunoglobulin capable of binding an antigen, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0025] As used herein, the term “specifically binding” is synonymous with “specifically recognizing” and means that an antigen and an antibody (or fragment thereof) specifically interact through an immunological reaction.

[0026] The present invention can also utilize an aptamer that specifically binds to the DNp63α protein instead of an antibody. As used herein, the term "aptamer" refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. General information on aptamers is described in detail in Hoppe-Seyler F, Butz K, "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R, "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7(1998).

[0027] According to a specific embodiment of the present invention, the agent for measuring the expression level of the gene encoding the DNp63α protein is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

[0028] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0029] The term “primer” as used herein refers to an oligonucleotide that acts as an initiation point for synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), i.e., the presence of nucleotides and a polymerization agent such as DNA polymerase, and conditions of suitable temperature and pH. Specifically, the primer is a single chain of deoxyribonucleotides. The primer used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and may also include ribonucleotides.

[0030] The primer of the present invention may be an extension primer that anneals to a target nucleic acid and forms a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which extends to a position to which an immobilized probe is annealed and occupies a portion to which the probe is annealed.

[0031] The extension primer used in the present invention includes a hybridizing nucleotide sequence complementary to a specific base sequence of a target nucleic acid, for example, a DNp63α-encoding gene. The term “complementary” means that the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under predetermined annealing or hybridization conditions, and includes both substantially complementary and perfectly complementary sequences, and specifically means perfectly complementary. As used herein, the term “substantially complementary sequence” includes not only a completely identical sequence, but also a sequence that is partially mismatched with the sequence to be compared, within a range that can anneal to a specific sequence and serve as a primer.

[0032] The primer must be sufficiently long to prime the synthesis of the extension product in the presence of a polymerization agent. The appropriate primer length depends on several factors, such as temperature, pH, and the primer source, but is typically 15-30 nucleotides. Shorter primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The design of such primers can be readily accomplished by those skilled in the art, using the target nucleotide sequence as a reference, and can be accomplished, for example, using a primer design program (e.g., PRIMER 3).

[0033] As used herein, the term “probe” refers to a linear oligomer having a natural or modified monomer or linkage comprising deoxyribonucleotides and ribonucleotides that can hybridize to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, and more specifically, is a deoxyribonucleotide. As the probe used in the present invention, a sequence that is perfectly complementary to a specific base sequence of the TRIM40 gene may be used, but a sequence that is substantially complementary may also be used as long as it does not interfere with specific hybridization. In general, since the stability of a duplex formed by hybridization tends to be determined by the identity of the terminal sequence, it is preferable to use a probe that is complementary to the 3'-end or 5'-end of the target sequence.

[0034] Suitable conditions for hybridization can be determined by referring to those disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0035] According to a specific embodiment of the present invention, the breast cancer for which the subtype is to be determined using the composition of the present invention is a breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2). More specifically, it is triple-negative breast cancer (TNBC) that does not express ER, PR, and HER2.

[0036] According to a specific embodiment of the present invention, when the expression of DNp63α is detected, the breast cancer is determined to be an inflammatory and epithelial-mesenchymal transition (EMT) activated subtype.

[0037] As shown in the examples described below, patient-derived triple-negative breast cancer organoids were classified into DNp63α-expressing and DNp63α-non-expressing groups, and pathway enrichment analysis based on DEG (Differentially Expressed Gene) showed that the DNp63α-expressing group had amplified NFκB-mediated TNF-α signaling pathway, epithelial-mesenchymal transition pathway, and inflammatory response pathway compared to the DNp63α-non-expressing group, confirming that DNp63α can function as a marker of a subtype with stronger inflammation, invasiveness, and metastasis.

[0038] According to a more specific embodiment of the present invention, the inflammatory and epithelial-mesenchymal transition activation subtypes have sensitivity to apoptosis inducers.

[0039] As shown in the examples described below, it was observed that apoptosis was specifically induced in the DNp63α expression group when DNp63α was inhibited.

[0040] In this specification, the term “apoptosis inducing agent” refers to a pharmacological ingredient that directly induces the death of cancer cells by activating apoptosis in cancer cells. Unlike necrosis, which is a passive cell death process, apoptosis, which is an active suicide mechanism, forms cysts called apoptotic bodies through cell shrinkage, nuclear condensation, and DNA fragmentation, and are removed by phagocytosis without an inflammatory response. Endogenous factors that artificially induce apoptosis include cytokines such as TNF (tumor necrosis factor) α, FAS, and TRAIL (TNF-related apoptosis inducing ligand), while exogenous factors include low-molecular-weight compounds such as capsaicin, actinomycin D, etoposide, and thapsigargin.

[0041] According to a specific embodiment of the present invention, if the expression of DNp63α is not detected, the breast cancer is determined to be a proliferative subtype.

[0042] As shown in the examples described below, the DNp63α non-expressing group showed amplified G2M checkpoint and E2F targets compared to the DNp63α expressing group, and the proliferation markers ki67 and EdU were specifically highly expressed, confirming that they can function as markers of a subtype with very strong proliferation.

[0043] According to another aspect of the present invention, the present invention provides a composition for predicting therapeutic response to an apoptosis-inducing agent in a breast cancer patient, the composition comprising, as an active ingredient, a preparation for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding the same.

[0044] The apoptosis inducer used in the present invention and the type of breast cancer for which treatment responsiveness is to be predicted have already been described above, so their description is omitted to avoid excessive duplication.

[0045] According to another aspect of the present invention, the present invention provides a composition for preventing or treating breast cancer, comprising an inhibitor for Delta Np63 alpha (DNp63α) protein as an active ingredient.

[0046] The term “inhibitor” as used herein means a substance that causes a decrease in the activity or expression of DNp63α protein, not only such that the activity or expression of DNp63α becomes undetectable or is present at an insignificant level, but also such that the activity or expression of DNp63α is reduced to such an extent that apoptosis of breast cancer cells can be significantly promoted.

[0047] The term “reduction in expression” in this specification may mean a state in which the expression level of DNp63α is reduced by, for example, 20% or more compared to the control group, more specifically, 30% or more, and even more specifically, 40% or more.

[0048] As used herein, the term “reduction in activity” refers to a measurable and significant decrease in the intrinsic function of DNp63α in vivo compared to a control, and specifically refers to a decrease in the activity of DNp63α to the extent that apoptosis of breast cancer cells can be significantly promoted. The decrease in activity includes not only a simple decrease in function but also ultimate inhibition of activity due to a decrease in stability.

[0049] Inhibitors of DNp63α include, but are not limited to, shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids), antisense oligonucleotides, CRISPR systems that suppress the expression of DNp63α proteins, the amino acid sequence and encoding nucleotide sequence of which are already known in the art, at the gene level, guide RNAs that recognize target genes, antibodies or aptamers that suppress at the protein level, as well as small molecule compounds, peptides, and natural products that suppress their activity, and all possible suppression means at the gene and protein levels can be used.

[0050] According to a specific embodiment of the present invention, the inhibitor for the DNp63α protein is an antibody or an antigen-binding fragment thereof that specifically binds to the DNp63α protein; or a nucleic acid molecule that inhibits the expression of a nucleotide encoding the DNp63α protein.

[0051] According to the present invention, the DNp63α inhibitor of the present invention may be a DNp63α-specific antibody that inhibits the activity of DNp63α at the protein level. The antibody that specifically recognizes DNp63α is a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody.

[0052] The term “nucleic acid molecule that suppresses expression” as used herein means a nucleic acid molecule that can specifically recognize a target gene by including a complementary nucleic acid sequence that can hybridize with the target gene and cause a modification in the nucleotide structure that causes a decrease in its function, and includes, for example, the shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, and gRNA included in the CRISPR system described above.

[0053] As used herein, the term “complementary” means that a nucleic acid molecule for suppressing expression is sufficiently complementary to a target nucleic acid sequence to selectively hybridize under certain annealing or hybridization conditions, and has a meaning that includes both substantially complementary and perfectly complementary, and preferably means perfectly complementary. As used herein, the term “substantially complementary sequence” includes not only a completely identical sequence, but also a sequence that is partially mismatched with the sequence to be compared, within the range where sequence-specific hybridization can occur by annealing to a specific sequence.

[0054] As used herein, the term “shRNA (small hairpin RNA)” refers to an RNA sequence that forms a tight hairpin structure to suppress the expression of a target gene through RNA interference, which is a single-stranded structure consisting of 50-70 nucleotides that forms a stem-loop structure in vivo. Typically, a long RNA of 19-29 nucleotides complementarily base-pairs on both sides of a loop region of 5-10 nucleotides to form a double-stranded stem, and is transduced into cells through a vector containing a U6 promoter to ensure constant expression, and is usually passed on to daughter cells to ensure heritable suppression of the expression of the target gene.

[0055] The term “siRNA” in this specification refers to a short double-stranded RNA that can induce RNAi (RNA interference) by cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and either blunt-ended or cohesive-ended is possible as long as it can suppress the expression of the target gene through the RNAi effect. The cohesive-ended structure can be either a 3-terminal protruding structure or a 5-terminal protruding structure.

[0056] In this specification, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is an oligonucleotide that is not expressed in cells and has a short stem-loop structure and suppresses target gene expression through complementary binding to the mRNA of the target gene.

[0057] As used herein, the term "ribozyme" refers to an RNA molecule that functions like an enzyme, recognizing a specific base sequence in RNA and cleaving it. A ribozyme consists of a region that specifically binds to a complementary base sequence of a target mRNA strand and a region that cleaves the target RNA.

[0058] As used herein, the term "PNA (Peptide Nucleic Acid)" refers to a molecule that possesses properties of both nucleic acids and proteins and can complementarily bind to DNA or RNA. PNA is not found in nature and is artificially synthesized through chemical methods. It forms a double strand through hybridization with a natural nucleic acid of complementary base sequence, thereby regulating the expression of target genes.

[0059] As used herein, the term “antisense oligonucleotide” refers to a nucleic acid molecule that is a nucleotide sequence complementary to a sequence of a specific mRNA and binds to the complementary sequence in the target mRNA, thereby inhibiting its translation into protein, translocation into the cytoplasm, maturation, or any other essential activity for its overall biological function. Antisense oligonucleotides can be modified at one or more base, sugar, or backbone positions to enhance their potency (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatoms, heterocyclic sugar sulphonates, etc.

[0060] As used herein, the term "gRNA (guideRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized region. A representative example of such gene editing systems is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0061] The nucleic acid molecule of the present invention described above can suppress the expression of DNp63α at the genetic level by expressing it in triple-negative breast cancer patients. As used herein, the term "express" means artificially introducing a gene vector to cause a subject to express an exogenous gene or to increase the natural expression level of an endogenous gene, thereby making the gene replicable as an extrachromosomal element or by completion of chromosomal integration within the subject's cells. Accordingly, the term "expression" has the same meaning as "transformation," "transfection," or "transduction."

[0062] As used herein, the term "gene delivery system" refers to any means of transporting genes into cells, and gene delivery is synonymous with transduction of genes into cells. At the tissue level, the term "gene delivery" is synonymous with gene spread. Therefore, the gene delivery system of the present invention can be described as both a gene penetration system and a gene spread system.

[0063] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0064] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. When the composition of the present invention is administered to a subject, it promotes apoptosis of cancer cells in triple-negative breast cancer tissues by suppressing the activity or expression of the DNp63α protein, thereby suppressing the development of symptoms caused by the tumor, eliminating them, or alleviating them. Therefore, the composition of the present invention may be a composition for treating these diseases on its own, or may be administered together with other pharmacological ingredients and used as an adjuvant treatment for the diseases. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0065] As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0066] In the present invention, the term “therapeutically effective amount” means the content of a composition in which the pharmacological ingredient in the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a “prophylactically effective amount”.

[0067] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0068] According to a specific embodiment of the present invention, the breast cancer is a breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2). More specifically, it is triple-negative breast cancer (TNBC) that does not express ER, PR, and HER2, and most specifically, it is positive for the DNp63α protein. Therefore, the breast cancer that can be prevented or treated with the composition of the present invention may be ER(-) / PR(-) / HER2(-) / DNp63α(+) breast cancer that does not express ER, PR, and HER2, but expresses DNp63α.

[0069] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

[0070] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0071] The pharmaceutical composition of the present invention can be administered orally or parenterally, and specifically, can be administered orally, intravenously, subcutaneously, or intraperitoneally.

[0072] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0073] The pharmaceutical composition of the present invention may be formulated in a unit dose form or placed in a multi-dose container by using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or stabilizer.

[0074] According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating breast cancer, comprising the following steps:

[0075] (a) contacting a candidate substance with a biological sample containing cells expressing Delta Np63 alpha (DNp63α) protein; and

[0076] (b) a step of measuring the activity or expression level of DNp63α protein in the sample;

[0077] If the activity or expression level of the above DNp63α protein is reduced, the candidate substance is determined to be a composition for preventing or treating breast cancer.

[0078] The types of breast cancer that can be prevented or treated with the composition screened by the method of the present invention have already been described above, so their description is omitted to avoid excessive duplication.

[0079] The term "biological sample" as used herein refers to any sample containing cells expressing DNp63α obtained from a mammal, including a human, including, but not limited to, tissues, organs, cells, or cell cultures. Specifically, the biological sample may be a sample derived from a triple-negative breast cancer patient.

[0080] The term “candidate substance” used when referring to the screening method of the present invention means an unknown substance used in the screening to examine whether it affects the activity or expression level of DNp63α when added to a sample containing cells expressing DNp63α. The test substance includes, but is not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of DNp63α in a biological sample treated with the test substance can be performed by various expression level and activity measurement methods known in the art. As a result of the measurement, if the expression level or activity of DNp63α is decreased, the test substance can be determined as a composition for the prevention or treatment of breast cancer, specifically, triple-negative breast cancer, and most specifically, ER(-) / PR(-) / HER2(-) / DNp63α(+) breast cancer.

[0081] In the present invention, the term “determination device” means equipment capable of determining a disease, specifically a subtype of cancer, outside the body based on substances produced in the human body such as blood, saliva, urine, etc., and includes, for example, a measuring unit; a calculating unit; a detection unit; a determination unit; and an output unit, and is not limited as long as it is in the form of analyzing genes or proteins from the above substances.

[0082] The cancer subtype determination device of the present invention may be specifically a breast cancer subtype determination device, and may not only determine the cancer subtype of a subject, but may also be used to predict cancer prognosis for the subject, whether to receive chemotherapy, treatment responsiveness to chemotherapy, or prognosis after chemotherapy.

[0083] According to a specific embodiment of the present invention, a breast cancer subtype determination device is provided, comprising: a measuring unit for measuring the expression level of DNp63α protein or a gene encoding the same; and a determination unit for measuring the expression level of DNp63α and determining the subtype of breast cancer based on the measured result.

[0084] The agent used in the measuring unit of the above-described determination device of the present invention may be an agent for measuring the expression level of the DNp63α protein or a gene encoding the same. More specifically, the agent may include, but is not limited to, one or more selected from the group consisting of an antibody that specifically binds to the protein, or an antigen-binding fragment thereof; or an aptamer that specifically binds to the DNp63α protein.

[0085] By measuring the level of protein or gene expression using the preparation in the measuring unit of the present invention, breast cancer, more specifically, a subtype of breast cancer, can be determined.

[0086] The above-described judgment device of the present invention may further include a judgment unit that determines a subtype of breast cancer from the expression level of the protein or gene obtained from the measurement unit.

[0087] In the present invention, the judgment unit can determine the subtype of breast cancer based on the expression of the protein or gene obtained from the measurement unit.

[0088] In one example of the present invention, the judgment unit is characterized in that, if the expression of DNp63α measured by the measurement unit is detected, the breast cancer is judged to be an inflammatory and epithelial-mesenchymal transition (EMT) activated subtype, and if the expression of DNp63α is not detected, the breast cancer is judged to be a proliferative subtype.

[0089] According to a specific embodiment of the present invention, a method for preventing or treating breast cancer is provided, comprising a step of administering a pharmaceutical composition comprising an inhibitor for Delta Np63 alpha (DNp63α) protein as an active ingredient.

[0090] In the above embodiment, the method is provided, wherein the breast cancer is a breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

[0091]

[0092] In a specific embodiment of the present invention, a method for determining a subtype of breast cancer is provided, comprising the step of measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding the same.

[0093] In the above embodiment, the method for determining a subtype of breast cancer further includes a step of determining the breast cancer as an inflammatory and epithelial-mesenchymal transition (EMT) activation subtype if the expression of DNp63α is detected, and determining the breast cancer as a proliferative subtype if the expression of DNp63α is not detected; and the method provides a method in which the breast cancer is a breast cancer in which at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2) are not expressed.

[0094] In a specific embodiment of the present invention, the use of an agent for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding it is provided in determining the subtype of breast cancer, the use of an agent for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding it is provided in predicting the treatment response of a breast cancer patient to an apoptosis inducer, and the use of an inhibitor for Delta Np63 alpha (DNp63α) protein is provided in the prevention or treatment of breast cancer.

[0095]

[0096] The features and advantages of the present invention are summarized as follows:

[0097] (a) The present invention provides a composition for determining the subtype of triple-negative breast cancer and a composition for preventing or treating triple-negative breast cancer.

[0098] (b) By measuring the expression level of DNp63α, a biomarker discovered in the present invention, the molecular subtypes of triple-negative breast cancer can be accurately classified, and the treatment response to a specific therapeutic agent can be predicted with high reliability based on the classified molecular subtypes, ultimately contributing greatly to improving patient survival rates through early establishment of a treatment strategy.

[0099] (c) The present invention also significantly induces apoptosis of cancer cells by suppressing the expression of DNp63α in triple-negative breast cancer expressing DNp63α, and thus can be usefully used as an effective therapeutic composition for triple-negative breast cancer, which is an intractable solid cancer.

[0100]

[0101] Figure 1 is a diagram showing the results of unbiased clustering of patient-derived triple-negative breast cancer organoids through RNA sequencing analysis.

[0102] Figure 2a is a diagram showing 2,171 genes (DEGs) with different expression patterns in two subgroups (Group 1 and Group 2) of triple-negative breast cancer organoids based on the unbiased clustering results. Genes highly expressed in Group 1 are indicated in blue, and genes highly expressed in Group 2 are indicated in red. Figure 2b is a diagram showing the results of DEG-based pathway enrichment analysis. Figure 2c is a diagram showing the results of 3D immunofluorescence analysis for ki67 and EdU to compare the proliferation characteristics between the two groups.

[0103] Figure 3 is a diagram showing the difference in expression levels of basal markers in two subgroups (group 1 and group 2) of triple-negative breast cancer organoids through a heat map (Figure 3a) and Western blot (Figure 3b), respectively.

[0104] Figure 4 shows the results of a Western blot confirming the expression patterns of TP63 gene subtypes in two subgroups of organoids.

[0105] Figure 5 is a drawing showing the results of 3-D immunofluorescence staining using p63 antibody (4A4) in patient-derived triple-negative breast cancer organoids.

[0106] Figure 6 shows the results of a TUNEL assay to determine whether apoptosis was induced by TNF-α treatment (Figure 6a) and whether apoptosis was induced after treatment with DNp63-α targeting siRNA (Figure 6b) in two subgroups of triple-negative breast cancer organoids.

[0107] Figure 7 shows the results of comparing the changes in apoptosis gene expression following inhibition of DNp63-α expression in two subgroups of triple-negative breast cancer organoids.

[0108] Figure 8 is a drawing showing the results of comparing the degree of cell death according to DNp63-α targeting in triple-negative breast cancer 2-D cell lines through changes in cell morphology.

[0109] Figure 9 shows the results of comparing the degree of cell death according to DNp63-α targeting in triple-negative breast cancer 2-D cell lines using PI (propidium iodide) positivity.

[0110] Figure 10 is a drawing showing the results of grouping patient groups based on the results of DNp63-α immunofluorescence staining in surgical tissue (FFPE) samples from triple-negative breast cancer patients.

[0111]

[0112] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0113]

[0114] Example

[0115] Establishment of patient-derived organoids

[0116] Surgical biopsy tissues from TNBC patients provided by Seoul National University Hospital were mechanically minced using a blade and digested in dissociation solution [Advanced DMEM / F-12 (12634-010; Gibco) containing 1 mg / ml collagenase P (11213865001; Roche), 0.1 mg / ml DNase I (M0303S; Biolabs)] for 20 min. The samples were then washed twice in DMEM / F-12 medium to remove floating lipids and embedded in growth factor-reduced Matrigel (354230; Corning) in 12-well culture plates (cv01012; Celvest). Organoids were cultured in an incubator at 37°C and 5% CO2 using organoid culture medium.

[0117]

[0118] Components of organoid culture medium

[0119] Advanced DMEM / F-12 (12634-010; Gibco), 10 mM HEPES (15630-080; Gibco), 100 U / ml penicillin-streptomycin (15140122; Thermofisher), 1X GlutaMAX™ Supplement (35050061; Gibco), primocin (ant-pm-05; Invivogen), 10% R spondin-1 conditioned medium (in-house), 5 ng / ml human EGF (AF-100-15; Peprotech), 100 ng / ml Recombinant human Noggin (120-10C; Peprotech), 5 ng / ml recombinant human KGF (FGF-7) (100-19; Peprotech), recombinant human FGF-10 (100-26; Peprotech), 1X B-27™ Supplement (12587010; Gibco), 1.25 mM N-acetyl-L-cysteine ​​(A9165-5G; Sigma), 500 nM A83-01 (2939; Tocris), 500 nM SB202190 (S7067; Sigma), 5 mM nicotinamide (N3376; Sigma), 37.5 ng / ml human recombinant heregulin β-1 (100-03; Peprotech), 5 μM Y-27632 (72304; STEMCELL technologies).

[0120] *R spondin-1 conditioned medium was prepared in-house using the R spondin-1-secreting 293T cell line provided by the Hans Clevers Institute.

[0121]

[0122] 3-D immunofluorescence staining

[0123] Cultured organoids were detached from Matrigel using Recovery solution (354253; Corning) and washed twice with PBS. The organoids were transferred to a Matrigel-free microfluidic chip (H389600; Stemfit) and fixed with 4% PFA for 1 h. The organoids were permeabilized with 1% PBS-T (1% Triton X-100 in PBS) and washed twice with 0.2% PBS-T (0.2% Triton X-100 in PBS). The cells were then incubated with primary antibodies at a 1:500 dilution in blocking solution (3% BSA in 0.2% PBS-T) for 1 h, followed by treatment with secondary antibodies at a 500:1 dilution overnight. After washing with PBS, the cells were treated with 5 μg / ml DAPI solution (PBS base) for 3 h, and images were acquired using a confocal microscope (Zeiss, LSM-700). The antibodies used were purchased from the following sources: ER (ab75635; abcam), PR (PR-AT4.14; Invitrogen), HER2 (AF1129-SP; R&D systems), CD44 (ab189524; abcam), CD24 (ab134375; abcam), ALDH1 (PA5-32127; Invitrogen), Ki67 (ab-15580; abcam), p65 (sc-109; Santa Cruz).

[0124]

[0125] Bioinformatics analysis

[0126] In this study, quality confirmation was performed using the NGS QC Toolkit (version 2.3.3). Reads were then aligned to the hg19 reference using Mapsplice (version 2.2.1). Gene detection and subtype expression levels were then measured from the aligned data using RSEM (version 1.3.3). Differential gene expression analysis was performed using DESeq2 (version 1.30.1). DESeq2 can identify genes with statistically significant differential expression. Furthermore, functional enrichment analysis was performed using fgsea (version 1.16.0), a tool that aids in the interpretation of gene lists by identifying overrepresented biological terms within various gene categories. Through the integrated use of DESeq2 and fgsea, we aimed to elucidate the molecular mechanisms underlying the observed transcript abundance changes in this study.

[0127] Gene expression heatmaps were generated using Morpheus software (Morpheus, https: / software.broadinstitute.org / morpheus)

[0128]

[0129] Western blot analysis

[0130] Proteins were extracted using NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-Cl, pH 8.0, 0.5% NP-40, and protease inhibitors), and 50 μg of protein was loaded. Antibodies for each protein were obtained from the following sources: p63 (Clone 4A4), CK-14 (ab51054; abcam), EGFR (06847; Millipore), and GAPDH (2118; Cell signaling).

[0131]

[0132] siRNA treatment

[0133] The siRNAs used in the present invention are as follows: siLuciferase (SP-3003, Bioneer); sip63#1, 5'-AACCAUGAGCUGAGCCGUGAA-3' (SEQ ID NO: 1, Bioneer); sip63#2, 5'- CCACUGAACUGAAGAAACU-3' (SEQ ID NO: 2, Bioneer). Lipofectamine™ RNAiMAX (13778075; Invitrogen) was used as a transfection reagent.

[0134]

[0135] EdU staining

[0136] Organoids in culture medium were treated with 2 μM EdU for 16 h, collected, and washed with PBS. Subsequent procedures were performed in the microdissection chip in the same manner as in 3-D immunofluorescence staining. Organoids were fixed with 4% PFA, washed three times with PBS, and incubated in a staining mixture (2 mM CuSO4, 20 mg / ml ascorbic acid, 1:500 Sulfo-Cyanine5 azide (A3330; Lumiprobe) in PBS) for 30 min at room temperature. After washing three times with PBS, they were treated with a 5 μg / ml DAPI solution for 3 h, and confocal microscopy images were acquired at a wavelength of 647 nm (Zeiss, LSM-700).

[0137]

[0138] TUNEL assay

[0139] Cultured organoids were collected and fixed in a microfluidic chip with 4% PFX for 1 h at room temperature. The fixed organoids were washed with phosphate-buffered saline (PBS) and incubated in a permeabilization solution (1% PBS-T, 0.1% sodium citrate dissolved in PBS) for 1 h at room temperature. After washing twice with PBS, they were applied to the In Situ Apoptosis Detection Kit (11684795910; Roche). Images were generated using a confocal microscope (Zeiss, LSM-700) and the FITC channel.

[0140]

[0141] Quantitative RT-PCR

[0142] RNA was extracted from organoids using TRIzol™ (15596026; Invitrogen). cDNA was synthesized from 1 μg of RNA using the High Capacity cDNA Reverse Transcription Kit (4368814; AppliedBiosystems™), and qPCR was performed using PowerUp™ SYBR™ Green Master Mix according to the manufacturer's instructions. The primers used to detect gene expression were as follows: human Bax, sense 5'-TGACATGTTTTCTGACGGCAAC-3' (SEQ ID NO: 3), antisense 5'-GGAGGCTTGAGGAGTCTCACC-3' (SEQ ID NO: 4); Noxa, sense 5'-GAGATGCCTGGGAAGAAGG-3' (SEQ ID NO: 5), antisense 5'-ACGTGCACCTCCTGAGAAAA-3' (SEQ ID NO: 6); Human Puma, sense 5'-ACGACCTCAACGCACAGTACGAG-3' (SEQ ID NO: 7), antisense 5'-AGGAGTCCGCATCTCCGTCAGTG-3' (SEQ ID NO: 8). ΔNp63α, sense 5'-GGAAAACAATGCCCAGACTC-3' (SEQ ID NO: 9), antisense 5'-GTGGAATACGTCCAGGTGGC-3' (SEQ ID NO: 10). Bar graphs were generated using GraphPad Prism 5 software.

[0143]

[0144] Unbiased clustering of patient-derived triple-negative breast cancer organoids using RNA sequencing.

[0145] The present inventors aimed to determine whether consistent patterns of gene expression or biomarkers exist within established TNBC organoid lines through RNA-level analysis. To this end, RNA was extracted from 18 patient-derived triple-negative breast cancer organoids, RNA sequencing data was obtained, and mutation analysis was performed on 10 of these organoids. Using iDEP.95 software, a web application for RNA sequencing data, unbiased clustering of organoids based on the whole transcriptome revealed that TNBC organoids could be clearly distinguished into two subgroups exhibiting distinct gene expression profiles (Fig. 1).

[0146]

[0147] Comparison of two subgroups of triple-negative breast cancer organoids

[0148] We defined two subgroups, Group 1 and Group 2, through unbiased clustering and identified 2,135 differentially expressed genes (DEGs) between them. Of these, 754 genes were highly expressed in Group 1 and underexpressed in Group 2, and 1,381 genes were highly expressed in Group 2 and underexpressed in Group 1 (Fig. 2a).

[0149] Furthermore, we investigated the characteristics of each organoid subgroup using pathway enrichment analysis based on DEGs according to the Molecular Signatures Database (mSigDB) hallmark gene set (DEGs with |log2FC|>1 and t-test p value <0.05 were selected for analysis). As shown in Fig. 2b, group 1 organoids showed amplification of “G2M checkpoint” and “E2F target,” whereas group 2 organoids showed amplification of NFκB-mediated TNF-α signaling, epithelial-mesenchymal transition (EMT), inflammatory response, and the p53 pathway. This revealed that group 1 showed high expression of genes related to cell cycle and cell proliferation, whereas group 2 showed high expression of factors related to inflammation and EMT. This confirmed that Group 1 organoids were more proliferative than Group 2, while Group 2 was more inflammatory and invasive. Indeed, 3D immunofluorescence analysis of the proliferation markers ki67 and EdU revealed different degrees of proliferation between the two groups (Fig. 2c).

[0150]

[0151] Comparison of Basal Marker Expression in Two Subgroups of Triple-Negative Breast Cancer Organoids

[0152] The breast epithelium is composed of luminal cells and basal (myoepithelial) cells, and their characteristics vary depending on their origin. Characteristic genes expressed in these cells are associated with development, morphogenesis, and tumor behavior. In particular, the genetic characteristics of breast cancer and the resulting molecular subtypes are significantly associated with tumor aggressiveness, metastatic risk, and treatment responsiveness. We analyzed DEGs, focusing on luminal / basal markers differentially expressed in the two subgroups. Interestingly, several basal markers, such as KRT5, KRT6, EGFR, ITGA6, and TP63, showed high mRNA levels in Group 2, whereas luminal markers, such as KRT18, KRT19, and GATA3, showed no significant difference between the two groups (Fig. 3, left). Consistent with the mRNA data, Western blot results showed that basal markers, cytokeratin 14, EGFR, and p63, were highly expressed in Group 2 (Fig. 3, right).

[0153]

[0154] Confirmation of TP63 isoform expression in two subgroups of organoids

[0155] Western blotting confirmed that DNp63-α, a TP63 isoform, was specifically expressed in Group 2 (Fig. 4). p63, a member of the p53 family, is essential for normal epithelial development, regenerative proliferation, and differentiation. In particular, p63, a marker of epithelial stem cells, is highly expressed in the basal layer of stratified squamous epithelium, such as hair follicles and keratinocytes. p63 provides stemness, including proliferation, differentiation, and self-renewal, to maintain tissue homeostasis. During embryogenesis, p63 deficiency causes severe defects in squamous epithelium, skin, limb, and breast epithelial tissues. We confirmed that p63, a basal marker, was specifically expressed in Group 2 (Fig. 4). Given that p63 accompanies the expression of basal markers in various tissues and is a key regulator of breast tissue homeostasis, we hypothesized that p63 expression would determine the characteristics of TNBC organoid subgroups.

[0156] Furthermore, to identify the p63 isoform expressed in group 2 organoids, Western blot was performed using the 4A4 p63 antibody, which can detect various p63 isoforms. To distinguish the size, lysates from 293T cells transduced with plasmids expressing the p63 isoforms TAp63α and DNp63α (Delta-Np63-alpha) were loaded as a control. As a result, it was confirmed that group 2 organoids specifically expressed DNp63α, the major p63 isoform (Fig. 4).

[0157]

[0158] Patient differentiation through DNp63α immunofluorescence staining in triple-negative breast cancer organoids

[0159] By performing 3-D immunofluorescence staining using p63 antibody (4A4) in two groups of organoids and comparing the expression of DNp63α in each group, it was confirmed that DNp63α was expressed only in group 2 organoids, confirming that triple-negative breast cancer can be distinguished based on the expression level of DNp63-α (Fig. 5).

[0160]

[0161] Comparison of apoptosis induced by DNp63-α targeting

[0162] Following the confirmation of differences in DNp63-α expression levels in triple-negative breast cancer organoids, we sought to determine differences in cellular responses according to DNp63-α targeting. To this end, we examined whether apoptosis was induced by TNF-α treatment using a TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. As a result, unlike Group 1 organoids, Group 2 organoids showed positive TUNEL staining, indicating that apoptosis occurred in response to TNF-α only in Group 2 (Fig. 6a).

[0163] In order to compare three organoids from each subgroup, DNp63α was knocked down by treating with siRNA and apoptosis was confirmed through a TUNEL assay. As a result, TUNEL staining was positive only in group 2 organoids, which are the DNp63α expression group, confirming that apoptosis occurred specifically (Fig. 6b).

[0164]

[0165] Comparison of apoptotic gene expression according to DNp63α targeting

[0166] After knocking down the expression of DNp63α using siRNA in two subgroups of organoids, changes in the expression levels of proapoptotic genes Bax, Noxa, and Puma were confirmed through RNA extraction and quantitative RT PCR. As a result, the expression of the three genes was not induced at all in group 1 organoids when treated with TNF-α + CHX, whereas the expression of all these genes increased in group 2 organoids (Fig. 7). This indicates that the two TNBC organoid subgroups showed different responses to TNF-α-mediated DNp63α targeting.

[0167]

[0168] Comparison of apoptosis by targeting DNp63α in triple-negative breast cancer 2-D cell lines

[0169] We compared the effects of DNp63α-targeting on apoptosis using DNp63α-positive and DNp63α-negative triple-negative breast cancer cell lines. siRNA treatment confirmed that apoptosis occurred only in DNp63α-positive cell lines (Fig. 8).

[0170]

[0171] Comparison of apoptosis by DNp63-α targeting in triple-negative breast cancer 2-D cell lines

[0172] After siRNA treatment in DNp63α-positive and DNp63α-negative triple-negative breast cancer cell lines, apoptosis was confirmed using propidium iodide (PI). As a result, high PI positivity, i.e., cell death, was confirmed only in DNp63α-positive cell lines (Fig. 9).

[0173]

[0174] Patient differentiation through DNp63α immunofluorescence staining in surgical tissue (FFPE) samples from triple-negative breast cancer patients.

[0175] Regarding the biomarker DNp63α discovered through the organoid system, immunofluorescence staining was performed on organoids derived from the surgical tissues of the patients, and it was confirmed that the expression of DNp63α in the organoids was consistently observed in the surgical tissues of the patients, and thus triple-negative breast cancer patients could be classified based on the expression of DNp63α (Fig. 10).

[0176]

[0177] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for determining the subtype of breast cancer, comprising as an active ingredient a preparation for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding it.

2. In paragraph 1, A composition characterized in that the agent for measuring the expression level of the DNp63α protein is an antibody or an antigen-binding fragment thereof that specifically binds to the DNp63α protein; or an aptamer that specifically binds to the DNp63α protein.

3. In paragraph 1, A composition characterized in that the agent for measuring the expression level of the gene encoding the above DNp63α protein is a primer or probe that specifically binds to a nucleic acid molecule of the above gene.

4. In paragraph 1, A composition characterized in that the above breast cancer is breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

5. In paragraph 1, A composition characterized in that, when the expression of the above DNp63α is detected, the breast cancer is determined to be an inflammatory and epithelial-mesenchymal transition (EMT) activated subtype.

6. In paragraph 5, A composition characterized in that the above-mentioned inflammatory and epithelial-mesenchymal transition activation subtypes have sensitivity to an apoptosis inducer.

7. In paragraph 1, A composition characterized in that if the expression of the above DNp63α is not detected, the breast cancer is determined to be a proliferative subtype.

8. A composition for predicting the therapeutic response of breast cancer patients to an apoptosis-inducing agent, comprising as an active ingredient a preparation for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding the same.

9. In paragraph 8, A composition characterized in that the above breast cancer is breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

10. A composition for preventing or treating breast cancer, comprising an inhibitor of Delta Np63 alpha (DNp63α) protein as an active ingredient.

11. In paragraph 10, A composition characterized in that the inhibitor for the above DNp63α protein is an antibody or an antigen-binding fragment thereof that specifically binds to the DNp63α protein; or a nucleic acid molecule that inhibits the expression of a nucleotide encoding the DNp63α protein.

12. In paragraph 10, A composition characterized in that the above breast cancer is breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

13. In paragraph 12, A composition characterized in that the above breast cancer is positive for the DNp63α protein.

14. A method for screening a composition for preventing or treating breast cancer, comprising the following steps: (a) a step of contacting a candidate substance with a biological sample containing cells expressing Delta Np63 alpha (DNp63α) protein; (b) a step of measuring the activity or expression level of DNp63α protein in the sample; If the activity or expression level of the above DNp63α protein is reduced, the candidate substance is determined to be a composition for preventing or treating breast cancer.

15. In paragraph 14, A method characterized in that the above breast cancer is breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

16. In paragraph 15, A composition characterized in that the above breast cancer is positive for the DNp63α protein.

17. A device for determining the subtype of breast cancer, comprising a measuring unit for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding the same.

18. In paragraph 17, The above-mentioned judgment device determines that the breast cancer is an inflammatory and epithelial-mesenchymal transition (EMT) activated subtype when the expression of the above-mentioned DNp63α is detected. A determination device further comprising a determination unit characterized in that, if the expression of the above DNp63α is not detected, the breast cancer is determined to be a proliferative subtype.

19. A method for preventing or treating breast cancer, comprising administering a pharmaceutical composition containing an inhibitor of Delta Np63 alpha (DNp63α) protein as an active ingredient.

20. In paragraph 19, A method wherein the above breast cancer is a breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

21. A method for determining a subtype of breast cancer, comprising the step of measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding the same.

22. In paragraph 21, The method for determining the subtype of the above breast cancer is as follows: if the expression of the above DNp63α is detected, the breast cancer is determined to be an inflammatory and epithelial-mesenchymal transition (EMT) activated subtype; A method for determining a subtype of breast cancer, further comprising the step of determining that the breast cancer is a proliferative subtype if the expression of the above DNp63α is not detected.

23. In paragraph 21 or 22, A method wherein the above breast cancer is a breast cancer that does not express at least two markers selected from the group consisting of ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2).

24. Use of a preparation for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding it in determining the subtype of breast cancer.

25. Use of a preparation for measuring the expression level of Delta Np63 alpha (DNp63α) protein or a gene encoding it in predicting the therapeutic response to an apoptosis inducer in breast cancer patients.

26. Use of an inhibitor of Delta Np63 alpha (DNp63α) protein in the prevention or treatment of breast cancer.

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