KR102424255B1 - Use of SMP30 as a biomarker for diagnosis and malignancy assessment of human breast cancer and animal mammary cancer - Google Patents

Abstract

The present invention relates to the use of SMP30 (Senescence marker protein 30) for diagnosing human breast cancer and animal mammary cancer and malignancy diagnosis. The expression of SMP30 of the present invention is increased in human breast cancer and animal mammary cancer, and furthermore, the expression of SMP30 increases in proportion to the increase in the malignancy of the tumor. It can be quickly diagnosed, and furthermore, the malignancy of the tumor can be compared and diagnosed.

Description

Use of SMP30 as a biomarker for diagnosis and malignancy assessment of human breast cancer and animal mammary cancer

The present invention relates to a composition for diagnosing breast cancer or mammary gland cancer and evaluating the malignancy, a kit for diagnosis and malignancy evaluation including the same, and a method of providing information necessary for the diagnosis and malignancy evaluation of breast cancer or mammary gland cancer using the same.

Breast cancer is the cancer with the highest incidence and mortality among women, and its incidence is rapidly increasing, and it is known that about 600,000 women worldwide die from breast cancer every year. This is about 15% of the female population dying from cancer. As in humans, mammary gland cancer is the most common cancer in other animals and accounts for about 50-70% of tumors occurring in unneutered females. Although various treatments are being developed due to the development of medical technology, it is known that fast and accurate diagnosis is the most important for improving the prognosis and survival rate of breast and mammary cancer. However, when a malignant tumor is at an early stage, it is difficult to differentiate it from a benign tumor on a biopsy. Therefore, in order to confirm breast cancer or mammary gland cancer at an early stage, the help of diagnostic markers is needed. However, Her2, estrogen receptor, and progesterone receptor, which are known as conventional diagnostic markers, have very variable results in animal mammary cancer tissues, so there is a limit to their use as diagnostic markers for confirmation. Therefore, there are currently no diagnostic markers capable of simultaneously diagnosing human breast cancer and animal mammary cancer, and capable of evaluating the malignancy and prognosis of tumor cells.

On the other hand, SMP30 (Senescence marker protein 30) is known to play an important role in cell survival and protection, such as inhibiting oxidative damage and preventing apoptosis. However, on the other hand, there is a report that hepatocellular carcinoma can be diagnosed when the expression of SMP30 is decreased along with the gene encoding alcohol dehydrogenase (ADH) and the gene encoding antioxidant protein 2 (AOP2) in liver tissue, and further Studies have shown that SMP30 is underexpressed in human breast and prostate cancer as well. Therefore, it has been considered that the expression of SMP30 is reduced in cancer cells.

WO 2007/039550 A2

Contrary to the results of previous studies on SMP30, the present inventors considered that SMP30 is important for the survival of tumor cells, and hypothesized that the expression of SMP30 would increase as the proliferation and viability increased in malignant tumor cells. Accordingly, the present inventors investigated the expression pattern of SMP30 protein in human breast cancer and animal mammary gland cancer, and using this, a diagnostic marker that can be used in both human breast cancer and animal mammary cancer was developed and a tumor using the difference in the expression level of SMP30. We have come to complete the invention regarding the evaluation of the malignancy of cells.

Accordingly, an object of the present invention is to provide a composition for diagnosing breast cancer or mammary gland cancer, comprising an agent for measuring SMP30 (Senescence marker protein 30) protein or its mRNA expression level.

Another object of the present invention is to provide a composition for diagnosing malignancy of breast cancer or mammary gland cancer, comprising an agent for measuring SMP30 protein or mRNA expression level thereof.

Another object of the present invention is to provide a kit for diagnosing breast or mammary cancer or malignancy, comprising the composition according to the present invention.

Another object of the present invention is to provide a method for providing information necessary for diagnosing breast cancer or mammary gland cancer and malignancy.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a composition for diagnosing breast or mammary cancer, comprising an agent for measuring SMP30 (Senescence marker protein 30) protein or its mRNA expression level.

In addition, the present invention provides a composition for diagnosing malignancy of breast cancer or mammary gland cancer, comprising an agent for measuring the expression level of SMP30 protein or its mRNA.

In one embodiment of the present invention, the breast cancer or mammary gland cancer may occur in mammals including humans, but is not limited thereto.

In another embodiment of the present invention, the SMP30 may be one in which the expression of the tumorous adenomatous epithelial cells of breast cancer or mammary gland cancer is increased.

In another embodiment of the present invention, the agent for measuring the expression level of the mRNA may be a primer set or a probe that specifically binds to the mRNA, but is not limited thereto.

In another embodiment of the present invention, the agent for measuring the expression level of the protein may be an antibody or aptamer specific for the protein, but is not limited thereto.

In addition, the present invention provides a kit for diagnosing breast cancer or mammary gland cancer comprising the composition according to the present invention.

In addition, the present invention provides a kit for diagnosing the malignancy of breast cancer or mammary gland cancer, comprising the composition according to the present invention.

In addition, the present invention provides a method of providing information necessary for diagnosing breast cancer or mammary gland cancer, comprising measuring the expression level of SMP30 (Senescence marker protein 30) protein or its mRNA in a biological sample isolated from a subject.

In addition, the present invention provides a method for providing information necessary for diagnosing malignancy of breast cancer or mammary gland cancer, comprising measuring the expression level of SMP30 (Senescence marker protein 30) protein or its mRNA in a biological sample isolated from a subject. to provide.

In one embodiment of the present invention, the method for providing information necessary for diagnosing breast cancer or mammary gland cancer comprises the step of determining as breast cancer or mammary cancer when the SMP30 protein or its mRNA expression is increased compared to normal tissue or benign tumor It may further include, but is not limited thereto.

In another embodiment of the present invention, the method of providing information necessary for diagnosing the malignancy of breast or mammary cancer is when the expression of the SMP30 protein or its mRNA is increased compared to a control group knowing the malignancy level of the tumor, the The method may further include, but is not limited to, determining that the level of malignancy is higher than that of the control group.

In another embodiment of the present invention, the subject may be a mammal including a human, but is not limited thereto.

In another embodiment of the present invention, the biological sample is blood, whole blood, plasma, urine, saliva, tissue, cell, organ, bone marrow, microneedle aspiration sample, core needle biopsy sample, and vacuum suction biopsy It may be one or more selected from the group consisting of the specimen, but is not limited thereto.

In another embodiment of the present invention, the protein expression level is western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining method , may be measured by one or more methods selected from the group consisting of , immunoprecipitation assay, complement fixation assay, mass spectrometry, FACS, and protein chip, but is not limited thereto.

In another embodiment of the present invention, the mRNA expression level is one selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization and DNA chip. It may be measured by the above method, but is not limited thereto.

In addition, the present invention provides a use for diagnosing breast or mammary cancer or malignancy of an agent for measuring SMP30 (Senescence marker protein 30) protein or its mRNA expression level.

In addition, the present invention provides a method for diagnosing breast cancer or mammary gland cancer or malignancy, comprising measuring the SMP30 (Senescence marker protein 30) protein or mRNA expression level thereof.

The present invention relates to the use of SMP30 for diagnosing human breast cancer and mammary gland cancer in animals and evaluating the degree of malignancy. has the effect of comparative diagnosis.

1A and 1B are diagrams showing the expression pattern of SMP30 in canine normal mammary gland tissue, adenoma, and grade 1, grade 2 carcinoma: (FIG. 1a) SMP30 Microscopic image showing the results of immunohistochemical staining; ( FIG. 1B ) A graph comparing and quantifying the expression of SMP30 in benign adenoma and malignant carcinoma (*P < 0.05, **P < 0.01, mean±SD).
Figures 2a and 2b are diagrams showing the expression pattern of SMP30 in primary and secondary carcinomas of cats: (Fig. 2a) Microscopic images showing the results of immunohistochemical staining for SMP30; ( FIG. 2B ) A graph comparing and quantifying the expression of SMP30 in grade 1 and grade 2 carcinoma (*P < 0.05, **P < 0.01, mean±SD).
3a and 3b are diagrams showing the expression pattern of SMP30 in complex adenoma, benign mixed tumor, and carcinoma in dogs: ( FIG. 3a ) Immunohistochemical staining results for SMP30 Microscopy images shown; ( FIG. 3B ) A graph comparing and quantifying the expression of SMP30 in each tumor (*P < 0.05, **P < 0.01, mean±SD).
4 is a microscope image showing the results of immunofluorescence staining for the expression pattern of SMP30 in complex adenoma, benign mixed tumor, and carcinoma in dogs (magnification: X400).
5A to 5C are diagrams showing the expression patterns of SMP30, p-ERK, and ERK in normal genetic tissue, benign mixed tumor and complex carcinoma of dogs: ( FIG. 5A ) Immunohistochemical staining results A microscope image showing ; (FIG. 5B) immunoblotting results for SMP30; (FIG. 5c) Immunoblotting results for p-ERK and ERK.
6a and 6b are diagrams showing the expression patterns of pan-Cytokeratin and SMP30 in malignant mammary gland cancer (simple carcinoma) samples taken from dogs: ( FIG. 6a ) Microscopic images showing the results of immunohistochemical staining; (Fig. 6b) A microscope image showing the results of immunofluorescence staining (magnification: X400).
7 is a diagram showing the results of immunofluorescence staining for the expression patterns of vimentin, SMP30 and pan-Cytokeratin in primary cells collected from malignant mammary gland cancer (simple carcinoma) tissue from dogs (magnification: X400).
8A to 8C are diagrams illustrating immunoblotting results for the expression of SMP30, P-ERK and ERK in human breast cancer cell lines MCF-7 and MDA-MB-231: ( FIG. 8A ) Light microscopy of human breast cancer cell line image; (Fig. 8b) immunoblotting results for SMP30; (Fig. 8c) Immunoblotting results for p-ERK and ERK.
9 is a diagram showing the results of immunofluorescence staining for the expression of SMP30 in human breast cancer cell lines MCF-7 and MDA-MB-231 (magnification: X400).
10A and 10B are diagrams showing the expression pattern of SMP30 according to the malignancy (grade 1-3) in human breast cancer tissue: ( FIG. 10A ) Microscopic images showing the results of immunohistochemical staining; (FIG. 10b) A graph comparing the expression intensity of SMP30 in grade 2 and grade 3 carcinoma.

The present inventors observed the expression pattern of SMP30 (Senescence marker protein 30) in mammary gland tumors of humans and animals using human breast cancer tissues and breast cancer cell lines, mammary cancer tissues of dogs and cats, and primary cells of dogs, and the expression patterns were By verifying whether it is consistently reproduced regardless of species, it was confirmed that SMP30 can be used as a novel diagnostic marker capable of diagnosing breast cancer in humans as well as mammary cancer in animals such as dogs and cats, thereby completing the present invention.

In an embodiment of the present invention, as a result of confirming the expression of SMP30 by immunohistochemical staining, the expression of SMP30 in the mammary gland tissue of dogs increased as the malignancy of the neoplastic adenoepithelial cells increased (FIGS. 1a and 1b). . This expression pattern was also shown in the mammary gland cancer of cats (Figs. 2a and 2b). The expression of SMP30 was observed to be expressed only in neoplastic adenoepithelial cells among the two cells, adenoepithelial cells and myoepithelial cells constituting the mammary gland tissue. In immunohistochemical staining, SMP30 showed a positive reaction only in neoplastic glandular epithelial cells (FIGS. 3a and 3b), and in the immunofluorescence staining result, it was found that the expression site of SMP30 does not overlap with non-mentin, which targets mesenchymal-derived cells. was confirmed (see FIG. 4, Examples 1 and 2).

In another embodiment of the present invention, in the observation of protein expression through immunoblotting, similar to immunohistochemical staining and immunofluorescence staining, it can be observed that the expression of SMP30 increases as the malignancy of the neoplastic adenoid epithelial cells increases. and almost no expression of SMP30 was observed in normal mammary gland tissue and benign mammary gland tumors. In addition, it was observed that the expression pattern of ERK, a factor that promotes cell proliferation and survival, and inhibits apoptosis, matches the expression pattern of SMP30 (see FIGS. 5a to 5c, Example 3).

In another embodiment of the present invention, in order to retest the results in animal experiments, the expression of SMP30 was confirmed in malignant mammary gland cancer tissue and primary cells collected from the tissue ( FIGS. 6a , 6b and 7 ). As with the immunofluorescent staining method, the expression of SMP30 was observed only in neoplastic adenoid epithelial cells. The expression site of SMP30 was the same as that of pan-Cytokeratin, a marker for adenomatous epithelial cells, and the expression site did not overlap with vimentin, a mesenchymal-derived cell marker. In addition, it was confirmed again that the expression of SMP30 showed a strong positive reaction in malignant adenoid epithelial cells (see Example 4).

In another embodiment of the present invention, the expression of SMP30 was confirmed in breast cancer cell lines MCF-7 and MDA-MB-231 in order to retest the results in mammary cancer tissues of animals in human breast cancer ( FIGS. 8A to 8C and FIGS. 9 ). ), immunofluorescence staining and immunoblotting showed that the expression of SMP30 was increased in MDA-MB-231 with poor differentiation compared to MCF-7 with relatively good differentiation. In addition, it was observed that the expression pattern of this SMP30 is consistent with the ERK expression pattern, similar to the results in the canine mammary gland tissue (see Example 5).

In another embodiment of the present invention, the expression pattern of SMP30 in human breast cancer tissues was observed by immunohistochemical staining (FIGS. 10a and 10b), and the expression of SMP30 in human breast cancer tissues also increased as the malignancy of adenoid epithelial cells increased. It was confirmed that the increase (see Example 6).

Accordingly, the present invention may provide a composition for diagnosing breast cancer or mammary gland cancer and malignancy, including an agent for measuring SMP30 (Senescence marker protein 30) protein or mRNA expression level thereof.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for diagnosing breast cancer or mammary gland cancer, comprising an agent for measuring the expression level of SMP30 protein or its mRNA.

In addition, the present invention relates to a composition for diagnosing malignancy of breast cancer or mammary gland cancer, comprising an agent for measuring the SMP30 protein or mRNA expression level thereof.

As used herein, the term “SMP30 (Senescence marker protein 30)” is also referred to as “regucalcin”, and is known to play a multifunctional role in various cell types as a regulatory protein in an intracellular signaling system. The DNA sequence or amino acid sequence of the SMP30 or regucalcin is known, for example, human SMP30 is searched for as Q15493 in the protein database UniPort.

As used herein, the term “breast cancer” refers to cancer occurring in the breast, and may be used interchangeably with “mammary tumor”. For example, in humans it may be called breast cancer, and in dogs or cats it may be called mammary gland cancer. The breast cancer may include mammary gland breast cancer, lobule breast cancer, or a combination thereof. The breast cancer includes cancers arising from the ducts and epithelium of the lobes and cancers arising from the stroma.

As used herein, the term “protein” is used interchangeably with “polypeptide” or “peptide”, for example, refers to a polymer of amino acid residues as commonly found in proteins in their natural state. .

As used herein, the term “expression” refers to the production of a protein or nucleic acid in a cell.

As used herein, the term "protein" is used interchangeably with "polypeptide" or "peptide", for example, refers to a polymer of amino acid residues as commonly found in proteins in their natural state. . “Polynucleotide” or “nucleic acid” refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner analogous to naturally occurring nucleotides are also included. “mRNA” refers to RNA that transfers genetic information (gene-specific base sequence) to ribosomes that specify an amino acid sequence from a specific gene during protein synthesis.

As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a particular disease or disorder, determining whether a subject currently has a particular disease or disorder, a particular disease or disorder. determining the prognosis of a subject with a disease, or therametrics (eg, monitoring a subject's condition to provide information about the efficacy of treatment).

As used herein, the term “malignancy” refers to the grade of the tumor evaluated based on the polymorphism of the nucleus, the number of mitotic phases, the cell density, and the degree of invasion into the surrounding blood vessels and lymphatic vessels in the target tumor. Histologic malignancy for existing breast cancer is, for example, a semi-quantitative method for assessing histologic grades in breast carcinoma proposed by Elston & Ellis (1991). It can be determined by speculum examination with an optical microscope as a standard. According to the present invention, more accurate diagnosis of malignancy is possible by comparing the expression level of SMP30 with respect to the tumor tissue evaluated for malignancy by the conventional method.

In the present invention, the breast cancer or mammary gland cancer may occur in mammals including humans. The mammal may be, for example, a human, a dog, a cat, a mouse, a rabbit, a horse, a sheep, a hamster, a hedgehog, a ferret, or a guinea pig.

In the present invention, the SMP30 may be one in which the expression of the tumorous adenoepithelial cells of breast cancer or mammary gland cancer is increased.

When the composition of the present invention is for measuring the expression level of mRNA, the agent for measuring the expression level of mRNA may be a primer set or probe that specifically binds to mRNA.

The composition of the present invention comprising a primer set or probe specific for the SMP30 mRNA may further include an agent required for a known RNA detection method. Using the composition of the present invention, it is possible to measure the level of mRNA of the protein markers in a subject by using a known method for detecting RNA without limitation.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free three-terminal hydroxyl group, capable of base pairing with a complementary template and serving as a starting point for template strand copying. . Primers are capable of initiating DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.

As used herein, the term “probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled and thus specific mRNA existence can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.

The “primer” or “probe” may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, primers or probes can be variously modified according to methods known in the art within a range that does not interfere with hybridization with mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.

Meanwhile, in the present invention, the agent for measuring the expression level of the protein may be an antibody or aptamer that specifically binds to the protein.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, as long as it has antigen-antibody binding properties, a part of the whole antibody is also included in the antibody of the present invention, and all kinds of immunoglobulin antibodies that specifically bind to the protein of the present invention are included. For example, a functional fragment of an antibody molecule as well as a complete antibody having two full-length light chains and two full-length heavy chains, i.e. Fab, F(ab'), F(ab') with antigen-binding function. 2 and Fv, and the like. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies and chimeric antibodies and recombinant antibodies as long as they can specifically bind to the protein of the present invention.

As used herein, the term “aptamer” refers to a substance capable of specifically binding to an analyte to be detected in a sample, and a single-stranded nucleic acid (DNA, RNA, or modified) having a stable tertiary structure by itself. nucleic acid), it is possible to specifically confirm the presence of the target protein in the sample. According to the general aptamer preparation method, the aptamer is synthesized by determining the sequence of an oligonucleotide having a selective and high binding affinity to the target protein to be identified, and then the 5' or 3' end of the oligonucleotide is applied. To be able to bind to the functional group of the tamer chip, -SH, -COOH, -OH or NH ₂ It may be made by transformation, but is not limited thereto.

The composition of the present invention comprising an antibody specific for the SMP30 protein may additionally include an agent required for a method for detecting a known protein, and the method for detecting a known protein using the composition can be used without limitation. The expression level of a protein in a sample (in the present invention, a biological sample obtained from the subject) may be measured.

In the present invention, since the nucleic acid sequence of the gene encoding the protein marker is registered in the gene bank, those skilled in the art design an antisense oligonucleotide, primer set or probe that specifically amplifies a specific region of these genes based on the sequence. can do.

The antisense oligonucleotide can be chemically synthesized using the phosphoramidite solid support method or other well-known methods, and includes all functional equivalents. Also, the above-described “primer” or “probe” may be equally applied.

The term "functional equivalent" refers to, for example, one or more substitutions, deletions or additions from a reference sequence, the actual effect that does not result in various functional dissimilarities between the reference sequence and the subject sequence. (net effect) refers to both the nucleotide and nucleic acid sequences of the changed mutant sequence. Typically, such substantially equivalent sequences are only about 35% (i.e., the number of substitutions, additions, and deletions of each residue in the substantially equivalent sequence compared to the corresponding reference sequence and the remaining total number in the substantially equivalent sequence) is about 0.35 or less when divided by ), which varies from those listed herein. Such sequences have 65% sequence identity to the listed sequences. Substantially equivalent, eg mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity, more preferably at least 90% sequence identity with the listed amino acid sequences. Substantially equivalent nucleotide sequences of the invention may have a lower percentage of sequence identity, for example, given the redundancy or degeneracy of the genetic code. Preferably, the nucleotide sequence should have at least about 65% identity, more preferably at least about 75% identity, and most preferably about 95% identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent synthetic characteristics are treated as substantially equivalent. To determine equivalents, truncation of the mature sequence (eg, via a mutation that produces a spurious stop codon) should be neglected.

As another aspect of the present invention, the present invention may provide a kit for diagnosing breast cancer or mammary gland cancer comprising the composition according to the present invention. In addition, the present invention may provide a kit for diagnosing malignancy of breast cancer or mammary gland cancer, comprising the composition according to the present invention.

The kit of the present invention may include an antibody that recognizes a target protein as a marker or a primer set and probe that recognizes mRNA as a marker, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer set specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of the control gene. Other Reverse Transcription Polymerase Reaction Kits include test tubes or other suitable containers, reaction buffers (varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

In another specific embodiment, it may be a diagnostic kit comprising essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescent probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

As another aspect of the present invention, the present invention provides information necessary for diagnosing breast or mammary cancer, comprising measuring the expression level of SMP30 (Senescence marker protein 30) protein or its mRNA in a biological sample isolated from a subject can provide a way to

In the present invention, the method may further include, but is not limited to, determining as breast cancer or mammary gland cancer when the expression of the SMP30 protein or its mRNA is increased compared to normal tissues or benign tumors.

In addition, the present invention may provide a method for providing information necessary for diagnosing malignancy of breast cancer or mammary gland cancer, comprising measuring the expression level of SMP30 protein or its mRNA in a biological sample isolated from a subject.

In the present invention, when the expression of the SMP30 protein or its mRNA expression is increased compared to a control with a known malignancy grade of the tumor, the method may further include the step of determining that the malignancy is higher than that of the control group. However, the present invention is not limited thereto.

In the above step, if there is no control group that knows the malignancy grade of the tumor, the relative malignancy can be determined compared to a normal tissue or a benign tumor. For example, a person who intends to practice the present invention assigns an arbitrary malignancy grade to each section according to the expression increase rate (or multiple) of the SMP30 protein or its mRNA, and then selects the SMP30 protein or its mRNA from the sample isolated from the subject. can be quantified and determined as a malignancy grade corresponding to an increased ratio (or fold) compared to the expression level of SMP30 protein or its mRNA in normal tissues or benign tumors.

In the present invention, the subject may be a mammal including a human, and may be an individual suffering from or suspected of suffering from breast cancer or mammary gland cancer.

As used herein, the term “measurement” refers to measuring and confirming the presence (expression) of a target substance (marker protein in the present invention), or measuring a change in the presence level (expression level) of a target substance. And it is meant to include all of the confirmation. That is, measuring the expression level of the protein means measuring the expression (ie, measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and the experimental methods described herein are included therein. A specific protein level comparison method for each method is well known in the art. Therefore, the detection of the target protein is meant to include detection of the presence or absence of the SMP30 protein, or checking the increase (up-regulation) or decrease (down-regulation) of the protein expression level.

In the present invention, the biological sample may be used without limitation as long as it is collected from a subject for diagnosing breast cancer or mammary gland cancer or evaluating malignancy, for example, tissue, cells, blood, plasma, serum obtained by biopsy, etc. , saliva, nasal fluid, sputum, capsular fluid, amniotic fluid, ascites, cervical secretions, vaginal secretions, urine, cerebrospinal fluid, as well as biological samples such as microneedle aspiration samples. Preferably, at least one selected from the group consisting of a biological tissue sample or a cell sample, for example, a cell, tissue, organ, microneedle aspiration sample, a core needle biopsy sample, and a vacuum aspiration biopsy sample derived from a lesion tissue. can be measured in

The biological sample may be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. The sample may be prepared to increase the detection sensitivity of the protein marker, for example, a sample obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It may be pre-treated using a method such as sequential extraction or gel electrophoresis.

As used herein, the term “fine needle aspiration cytology (FNAC)” refers to the purpose of diagnosing benign or malignant cells after obtaining a small amount of cells by piercing a suspected breast tumor with a fine needle while viewing ultrasound. is implemented

The protein expression level measurement is not particularly limited as long as it is by a protein expression measurement method known in the art, but for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immune diffusion method (Ouchterlony). immunodiffusion), rocket immunoelectrophoresis, immunostaining, immunoprecipitation, complement fixation, mass spectrometry, FACS, and protein chip.

The gene expression level measurement is PCR, RNase protection assay, Northern blotting, Southern blotting, In situ hybridization, and DNA chip by a gene expression measurement method known in the art. It may be one or more selected from.

As used herein, the term “analysis” may preferably mean “measurement”, and the qualitative analysis may mean measuring and confirming the presence or absence of a target substance, and the quantitative analysis is It may mean measuring and confirming a change in the presence level (expression level) or amount of a desired substance. In the present invention, analysis or measurement may be performed without limitation, including both qualitative and quantitative methods, and preferably quantitative measurement may be performed.

The terms used in the present invention have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Throughout the specification of the present invention, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated. The terms "about", "substantially", etc. to the extent used throughout the specification of the present invention are used in or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and the present invention It is used to prevent unfair use by unconscionable infringers of the disclosure in which exact or absolute figures are mentioned to help the understanding of

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the examples.

[Example]

Experimental materials and methods

1. Sample Preparation

Human breast cancer tissues and cell lines and canine and cat mammary gland cancer tissues used in this experiment were selected to evaluate SMP30 expression and reproducibility in different species, and to observe the association of SMP30 expression according to malignancy. Human breast cancer tissues were used as tissue microarray (TMA) slides supplied by Biomax (USA), and mammary gland cancer tissues of dogs and cats were supplied and used from the Veterinary Hospital affiliated with Kyungpook National University College of Veterinary Medicine.

Primary cells were extracted from the resected mammary gland tissue and washed with DPBS (Dulbecco's Phosphate-Buffered Saline) buffer containing 1% penicillin-streptomycin. After finely cutting the mammary gland tissue, it was reacted with DPBS buffer containing 0.1% collagenase IV at 37° C. for 30 minutes and allowed to decompose the tissue. Thereafter, the cell suspension was filtered with a 40 μm cell strainer and the filtered cells were cultured. It was cultured using a growth medium, and cells after passage 5 were used in this experiment.

The test substance, SMP30 antibody, was used as a transparent liquid, supplied from CosmoBio (Japan). Reagents are methanol (Duksa, Korea), hematoxylin (Sigma, USA), eosin Y (Sigma), 3.7% paraformaldehyde (Sigma), 40% formaldehyde (Daejeong, Korea), toluene (Duksan, Korea) ), parafilm (Leica Histowax, Germany), etc. were used.

2. Histopathological observation

For histopathological examination of the excised dog and cat mammary gland cancer tissues, after fixing using 10% neutral formalin, embedding in paraffin through a general tissue processing process, making a tissue section with a thickness of 4 μm, drying it, Matoxylin-eosin (Hematoxylin-eosin) staining was observed with an optical microscope. Hematoxylin-eosin staining evaluated the malignancy of the mammary gland tissue. The malignancy of the tumor cells was evaluated based on the polymorphism of the nucleus, the number of mitotic phases, the cell density, and the invasion into the surrounding blood vessels and lymphatic vessels. On the other hand, human breast cancer tissue slides were provided by Biomax, and hematoxylin-eosin staining was performed to evaluate the degree of malignancy with an optical microscope.

3. Immunohistochemistry observation through immunohistochemistry

After fixing the tumor tissue in formalin, the prepared paraffin-embedded tissue was sectioned to a thickness of 4 μm, deparaffinized, and then treated with 3% H ₂ O ₂ at room temperature for 35 minutes to inhibit the activity of endogenous peroxidase. After washing with 0.01 M phosphate-buffered saline (PBS), in order to increase antigen expression in the tissue, 0.01 M citrate buffer was used in a microwave for 30 minutes. After washing 3 times with PBS, reacting with a blocking solution (Life Technologies, USA) for 1 hour, primary antibody SMP30 (1:200, Cosmo Bio Co., Ltd. Japan), pan-Cytokeratin ( 1:200, Abcam, USA) were treated and reacted overnight at 4°C. Then, washed 3 times for 5 minutes with PBS, treated with a broad spectrum secondary antibody (Life Technologies) and reacted for 60 minutes. After washing with a buffer solution, it was treated with HRP-conjugated streptavidin (hoseradish peroxidase-conjugated streptavidin) for 10 minutes, and then using a DAB (3,3-diaminobenzidine) peroxidase substrate kit (Vetor Laboratories, USA). was colored. After counterstaining with hematoxylin for 30 seconds, the degree of positive reaction to each antibody was observed under an optical microscope.

Human breast cancer TMA slides were incubated at 60° C. for 30 minutes for complete deparaffinization. After deparaffinization, 20 μg/mL proteinase K diluted in Tris-EDTA buffer was treated at 37° C. for 20 minutes to suppress non-specific reactions, followed by washing with PBS. The subsequent procedure was the same as the immunohistochemical staining and fixation performed on mammary gland cancer tissues in dogs and cats. The primary antibody against SMP30 was diluted 1:50 and stained.

4. Protein expression analysis by immunoblotting

Mammary cancer tissues of dogs stored frozen at -80°C were purified with 0.1 mM sodium vanadate (Na ₃ VO ₄ ) and protease inhibitor cocktail (Roche Diagnostics, Germany), Pefabloc (Roche Diagnostics), Sodium fluoride (sodium fluoride), sodium pyrophosphate (sodium pyrophosphate) was dissolved in the RIPA buffer was homogenized. The homogenized mammary cancer sample was centrifuged at 6,000 rpm and 4° C. for 10 minutes to remove fat, and then the supernatant was taken. The supernatant was again centrifuged at 13,300 rpm and 4°C for 20 minutes to obtain the supernatant.

Human breast cancer cell lines, MCF-7 and MDA-MB-231, contained 5 × 10 ⁵ fetal bovine serum (FBS; Invitrogen, USA) and penicillin-streptomycin-amphotericin B (WELGENE, After inoculation in DMEM (Dulbecco's modified Eagle medium; WELGENE, Korea) medium containing Korea), incubated at 37 ° C overnight, 0.1 mM sodium vanadate (Na ₃ VO ₄ ) and protease inhibitor cocktail purification (Roche Diagnostics) , Peppablock (Roche Diagnostics), sodium fluoride, and RIPA buffer in which sodium pyrophosphate was dissolved was harvested as a diluted solution of 1X electrophoresis sample buffer. Each sample was measured for protein concentration using a Bradford assay. Each protein sample was boiled at 100° C. for 10 minutes and then electrophoresed on a 10% SDS-polyacrylamide gel. For immunoblotting, the proteins in the electrophoresed gel were electro-transferred onto a PVDF membrane (Millipore, USA). After leaving for 1 hour in a 5% skim milk blocking solution dissolved in TBS (Tris-bufferd saline) containing 0.1% Tween-20, the primary antibody, anti-SMP30 antibody (1:2000, Cosmo Bio Co., Ltd.), anti-p-ERK antibody (1:1000, Santa Cruz Biotechnology, USA) and anti-ERK antibody (1:1000, Santa Cruz Biotechnology) were treated and reacted, respectively, and the same amount of protein In order to confirm this loading, anti-β-actin antibody (1:2000, Sigma, USA) was treated and reacted. After the reaction, the PVDF membrane was washed three times with TBS buffer in which 0.1% Tween-20 was dissolved, and the secondary antibody corresponding to the primary antibody was diluted in 5% skim milk at a ratio of 1:2000 and reacted at room temperature for 1 hour. . After washing sufficiently with TBS buffer, in order to observe a specific reaction, it was reacted with ProNATM ECL Ottimo (Translab, Korea) and exposed to Amersham ImagerTM 680 (GE Healthcare Bjorkgatan, Sweden).

5. Immunohistochemical observation through immunofluorescence staining

After fixing the tumor tissue in formalin, the prepared paraffin-embedded tissue was sectioned to a thickness of 4 μm, deparaffinized, and then treated with 3% H ₂ O ₂ at room temperature for 35 minutes to inhibit the activity of endogenous peroxidase. After washing with PBS, in order to increase antigen expression in the tissue, it was treated with 0.01 M citrate buffer using a microwave oven for 30 minutes. In the case of human breast cancer cell lines MDA-MB-231, MCF-7, and canine mammary cancer primary cells, 5 × 10 ⁴ were inoculated on the coverslips placed in advance in the wells of a 12-well plate and cultured overnight at 37°C. After that, it was fixed in 4% PFA for 20 minutes. Each pretreated sample was washed 3 times in PBS, and then treated with 0.1% Triton X-100 diluted in PBS for 10 minutes to increase tissue permeability to the antibody. After blocking with 5% donkey serum for 1 hour, the primary antibody, anti-SMP30 antibody (1:200, Cosmo Bio Co., Ltd.), and anti-pan-Cytokeratin antibody (1:200, Abcam) ), anti-vimentin antibody (1:200, Thermo Fisher Scientific, Inc., USA) were treated, respectively, and reacted at 4° C. overnight. Then, it was washed 3 times for 5 minutes with PBS, and reacted by treating the secondary antibody corresponding to the primary antibody for 60 minutes. After washing with a buffer solution and mounting with a DAPI (4',6-diamidino-2-phenylindole; Cell signaling, USA) solution, the degree of positive reaction to each antibody was observed through a fluorescence microscope.

Example 1. Confirmation of SMP30 expression pattern through immunohistochemical staining in mammary gland cancer tissues of dogs and cats

As a result of immunohistochemical staining using the SMP30 antibody in canine mammary cancer tissues, it was observed that the expression of SMP30 increased as the malignancy of tumor cells increased compared to normal mammary gland tissues ( FIGS. 1A and 1B ). In the case of benign mammary cancer tissues, the expression of SMP30 was similar or very insignificant to that of normal mammary gland tissues, and it was confirmed that there was a significant difference from the expression level of SMP30 observed in malignant mammary cancer tissues. On the other hand, the expression of SMP30 was observed only in proliferative neoplastic adenoid epithelial cells with abundant nucleolus, severe cell atypia, high cell density, and many mitotic phases. The same pattern was also observed in the feline mammary gland tissue ( FIGS. 2A and 2B ).

In order to accurately confirm the expression pattern of SMP30 as described above, as a result of immunohistochemical staining for SMP30 in mammary gland cancer tissues of dogs originating from cells derived from both adenoid epithelial cells and myoepithelial cells, the expression of SMP30 was found to be in the tumorous glands. It was confirmed only in epithelial cells, and the expression of SMP30 was not observed in myoepithelial cells. In addition, it was observed that the expression of SMP30 increases as the malignancy of the neoplastic adenomatous epithelial cells increases ( FIGS. 3A and 3B ).

Example 2. Confirmation of protein expression pattern through immunofluorescence staining of neoplastic adenoid epithelial cells

2.1. Confirmation of vimentin expression pattern

As a result of immunofluorescence staining using vimentin, an antibody that detects mesenchymal-derived cells such as myoepithelial cells, vimentin showed a positive reaction in proliferative myoepithelial cells, which are mesenchymal-derived cells, but not in neoplastic adenoepithelial cells. showed a negative reaction. In addition, this expression pattern showed a result in contrast to that SMP30 showed a positive reaction to the tumorous adenoid epithelial cells, but showed a negative reaction to the mesenchymal-derived myoepithelial cells (Fig. 4).

2.2. Confirmation of SMP30 expression pattern

Like immunohistochemical staining, it was confirmed that SMP30 showed a positive reaction only in neoplastic adenoepithelial cells, and the expression pattern increased in proportion to the malignancy of neoplastic adenoepithelial cells (FIG. 4).

Example 3. Analysis of Protein Expression in Canine Mammary Cancer Tissues

After protein was extracted from normal mammary gland tissue, benign mixed tumor and complex carcinoma tissue of dogs, immunoblotting was performed to analyze the expression pattern of the SMP30 protein ( FIGS. 5A to 5C ).

In the case of SMP30, the same pattern as the results of immunohistochemical staining and immunofluorescence staining was observed in the immunoblotting results. In contrast to the weak expression of SMP30 in normal mammary gland tissues and benign mixed tumors, it was observed that the expression of SMP30 was significantly increased in complex carcinoma, a malignant tumor ( FIG. 5b ). These results are considered to suggest that the expression of SMP30 is closely related to the proliferation and survival of tumor cells.

Meanwhile, as a result of immunoblotting for ERK, which is a mediator that inhibits apoptosis and promotes cell proliferation and survival, and p-ERK, which is an activated form of ERK, the same pattern as the expression pattern of SMP30 was observed. The expression of p-ERK was observed to be weak in normal mammary gland tissues and benign mixed tumors, but the expression of p-ERK was significantly increased in complex carcinoma with increased proliferative and malignant levels of adenoid epithelial cells. (Fig. 5c).

Example 4. Observation of protein expression in primary cells collected from canine malignant mammary cancer tissue

In order to revalidate the results confirmed in the mammary gland tissue in vitro, primary cells were extracted from malignant mammary gland cancer samples in dogs. A part of the excised tissue was subjected to H&E staining and immunohistochemical staining to observe histopathological findings (FIGS. 6a and 6b), and cells after culture and passage 5 after extracting primary cells from the remaining part was inoculated into a cover glass located in a 12-well plate as many as 5 × 10 ⁴ , and cultured for 24 hours, followed by immunofluorescence staining (FIG. 7).

4.1. Immunohistochemical staining results

Immunohistochemical staining was performed on the excised malignant mammary gland tissue for observation of histopathological findings. As a result, it could be observed that the expression patterns and expression positions of pan-Cytokeratin and SMP30, which target tumorous adenomatous epithelial cells, were consistent. Through this, it was confirmed that SMP30 was specifically expressed only in neoplastic adenomatous epithelial cells (FIG. 6a).

4.2. Immunofluorescence staining results

Based on the results of immunohistochemical staining performed previously, immunofluorescence staining was performed to specify the expression site of SMP30. As a result of immunofluorescence staining on the excised malignant mammary gland tissue, it was observed that the expression positions of SMP30 and pan-Cytokeratin were exactly the same as the results of immunohistochemical staining (FIG. 6b).

After confirming the expression pattern of SMP30 in the tissue, immunofluorescence staining was performed on primary cells collected and cultured from malignant mammary gland tissue in dogs. Immunofluorescence staining performed on primary cells also showed the same expression pattern as that observed in the canine malignant mammary gland tissue ( FIG. 7 ).

More specifically, it showed a negative reaction to vimentin, so it was possible to observe that the primary cells of the collected malignant mammary carcinoma were not mesenchymal-derived myoepithelial cells. In the case of SMP30, it was observed that a strong positive reaction was observed in the cytoplasm and nucleus of neoplastic adenoid epithelial cells. In addition, it could be observed that pan-Cytokeratin also showed a strong positive reaction to neoplastic adenoid epithelial cells, like SMP30, and it was confirmed that the collected primary cells were derived from a glandular epithelium (FIG. 7).

Example 5. Observation of protein expression in human breast cancer cell lines

In order to confirm whether the expression of SMP30 is equally reproduced in human breast cancer and in vitro scale, human breast cancer cell lines MCF-7 and MDA-MB-231 were used. MCF-7 is a breast cancer cell line with good differentiation, whereas MDA-MB-231 is known as a cell line with extremely poor differentiation ( Breast Cancer Research and Treatment . 1994; 31: 325-335). In fact, when these cells were cultured, MDA-MB-231 showed severe cell atypism, high cell density and short-lived nucleolus, whereas MCF-7 showed almost no cell atypism, moderate cell density and It showed an indistinct nucleolus (Fig. 8a). In these cells, the reproducibility of the expression pattern of SMP30 was confirmed through immunofluorescence staining and immunoblotting.

As a result of immunofluorescence staining, a similar expression pattern of SMP30 was observed in human breast cancer cell lines as in the mammary gland tissue of the animal. The expression of MCF-7 having a relatively good degree of differentiation was low, whereas in the case of MDA-MB-231 having a low degree of differentiation, it was observed that the expression of SMP30 was high ( FIG. 9 ).

Immunoblotting results also showed the same pattern as immunofluorescent staining. In the case of MCF-7 with good differentiation, SMP30 expression was hardly observed, whereas in the case of MDA-MB-231, the expression of SMP30 was increased (Fig. 8b), which was consistent with the expression pattern of p-ERK (Fig. 8c). ). These results are considered to mean that the expression of SMP30 in human breast cancer is closely related to the proliferation and survival of tumor cells.

Example 6. Confirmation of expression of SMP30 in human breast cancer tissue

Finally, the expression pattern of SMP30 in human breast cancer tissue was revalidated. SMP30 expression patterns were observed in human breast cancer tissues, similar to those of dog and cat mammary gland cancer tissues and human breast cancer cell lines. While almost no expression of SMP30 was observed in normal human breast tissue, expression of SMP30 was clearly observed in the neoplastic adenoid epithelium, and the expression level increased in proportion to the malignancy of the neoplastic adenoepithelial cells (Fig. 10a and 10b).

From all of the above examples, it could be observed that SMP30 was specifically expressed in tumorous adenoepithelial cells in both human breast cancer and mammary gland cancer of animals such as dogs and cats. , in particular, has been shown to be closely related to the proliferation and survival of tumor cells. The expression pattern of SMP30 is thought to play an important role in evaluating the malignancy and prognosis of tumors in the future human breast cancer and mammary gland cancer in dogs and cats. It is considered to be used as an essential marker.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (16)

A composition for diagnosing breast or mammary cancer, comprising an agent for measuring SMP30 (Senescence marker protein 30) protein or its mRNA expression level,
The SMP30 is characterized in that the expression is increased specifically in the tumorous adenoepithelial cells of breast cancer or mammary gland cancer, the composition.
According to claim 1,
The composition is characterized in that the composition for diagnosing malignancy of breast cancer or mammary gland cancer.
According to claim 1,
The breast or mammary gland cancer is characterized in that it occurs in mammals, including humans, the composition.
delete According to claim 1,
The agent for measuring the expression level of the mRNA is characterized in that the primer set or probe that specifically binds to the mRNA, the composition.
According to claim 1,
The agent for measuring the expression level of the protein is characterized in that the antibody or aptamer specific for the protein, the composition.
A kit for diagnosing breast cancer or mammary gland cancer, comprising the composition of any one of claims 1 to 3, and any one of claims 5 to 6.
8. The method of claim 7,
The kit is characterized in that it is a kit for diagnosing malignancy of breast cancer or mammary gland cancer.
A method of providing information necessary for diagnosing breast cancer or mammary gland cancer, comprising measuring the expression level of SMP30 (Senescence marker protein 30) protein or its mRNA in neoplastic adenoid epithelial cells in a biological sample isolated from a subject.
10. The method of claim 9,
The method further comprises the step of determining as breast cancer or mammary gland cancer when the expression of the SMP30 protein or its mRNA is increased compared to normal tissues or benign tumors,
The method, characterized in that the benign tumor comprises breast cancer or mammary gland cancer.
10. The method of claim 9,
The method, characterized in that for diagnosing the malignancy of breast cancer or mammary gland cancer.
12. The method of claim 11,
The method further comprises the step of determining that the level of malignancy is higher than that of the control, when the expression of the SMP30 protein or its mRNA is increased compared to a control with a known level of malignancy of the tumor. Way.
10. The method of claim 9,
The method, characterized in that the subject is a mammal including a human.
10. The method of claim 9,
The biological sample is at least one selected from the group consisting of blood, whole blood, plasma, urine, saliva, tissue, cell, organ, bone marrow, microneedle aspiration sample, core needle biopsy sample, and vacuum aspiration biopsy sample How to do it.
10. The method of claim 9,
The protein expression level was determined by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, mass Method, characterized in that measured by one or more methods selected from the group consisting of analysis (Mass spectrometry), FACS, and a protein chip.
10. The method of claim 9,
The mRNA expression level is PCR, RNase protection assay, Northern blotting (northern blotting), Southern blotting (southern blotting), in situ hybridization method, characterized in that measured by one or more methods selected from the group consisting of a DNA chip, Way.

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