Kit for diagnosing colorectal cancer senescent tumor cells comprising p15 protein or the gene encoding it

Abstract

The present invention relates to a biomarker composition for diagnosing senescent tumor cells of colorectal cancer comprising the p15INK4B protein or a gene encoding it. It is confirmed through analysis after inducing senescence in cells extracted from colorectal cancer patients that the expression of p15INK4B increases together with p16INK4A, and it is also confirmed in vitro that the expression of p15INK4B increases together with p16INK4A when cell senescence is induced. Therefore, p15INK4B can be utilized as a marker for diagnosing senescent tumor cells of colorectal cancer, and can be used in various ways such as a biomarker composition, a diagnostic composition, and a diagnostic kit.

Description

Technology Field

[0001] The present invention is p15 INK4B A biomarker composition for diagnosing senescent tumor cells of colorectal cancer is provided, comprising a protein or a gene encoding the same. Background Technology

[0002] With the recent Westernization of dietary patterns, the incidence of colorectal cancer has been increasing sharply and the age of onset is becoming younger; consequently, the need for early diagnostic tests such as colonoscopy, sigmoidoscopy, and colonography is growing. Factors that increase the incidence of colorectal cancer include a high-fat, low-fiber diet, certain genetic disorders such as familial polyposis, and inflammatory bowel diseases like ulcerative colitis. Unlike upper digestive tract cancer, symptoms of colorectal cancer often appear late, and even when symptoms do occur, they are frequently mistaken for simple constipation or hemorrhoids, leading to a missed opportunity for timely treatment.

[0003] In Korea, it is the fourth most common cancer in men, following stomach, lung, and liver cancer, and the third most common in women, following breast and stomach cancer. It has shown a continuous upward trend, accounting for 5.8% of all cancers in 1980, rising to 6.1% in 1985, 8.2% in 1995, and 12.3% in 2005. Compared to 1991 data, while mortality rates for stomach, liver, and cervical cancers decreased, the mortality rate due to colorectal cancer more than doubled, rising from 4.7 to 11.4 per 100,000 people. As is generally the case with most cancers, the symptoms of colorectal cancer are not specific to the colon. Therefore, it is difficult to identify specific symptoms as characteristic indicators of colorectal cancer. However, in general, colorectal cancer patients exhibit symptoms such as changes in bowel habits, bloody or mucous stools, thinning of stools, weight loss, abdominal discomfort (abdominal pain, bloating), fatigue, loss of appetite, vomiting, nausea, and anemia. As with all cancers, colorectal cancer can be cured if detected early and treated appropriately. However, in most cases, colorectal cancer is asymptomatic in its early stages, and when symptoms appear, the cancer has often already progressed significantly. This is because, in the early stages, the tumor is small and does not show any particular symptoms; however, as the tumor grows, it obstructs the passage of stool within the colon, and additionally, bleeding occurs from the tumor, and secretions are excreted from the surface of the tumor. Currently, screening tests used for colorectal cancer include fecal occult blood tests, tumor marker tests, barium enema, colonoscopy, computed tomography, abdominal ultrasound, transrectal ultrasound, and sigmoidoscopy; however, these methods have various limitations in early diagnosis. The actual survival rate is over 90% in stage 1, but drops below 5% in stage 4 when the disease has already spread to distant organs such as the lungs or liver.

[0004] Therefore, in order to increase the survival rate of colorectal cancer patients, research into methods for more accurate diagnosis is necessary. Prior art literature

[0005] 1. Oncogene volume 41, pages 943–959 (Published Oct. 19, 2021) The problem to be solved

[0006] The objective of the present invention is p15 INK4B The invention provides a biomarker composition for diagnosing colorectal cancer senescent tumor cells comprising a protein or a gene encoding the same.

[0007] Another objective of the present invention is p15 INK4B The invention provides a composition for diagnosing senescent tumor cells of colorectal cancer containing, as an active ingredient, a preparation capable of measuring the expression or activity level of a protein, or the expression level of a gene encoding said protein.

[0008] Another objective of the present invention is to provide a kit for diagnosing senescent tumor cells of colorectal cancer comprising the above composition.

[0009] Another objective of the present invention is to obtain p15 from a sample isolated from a colorectal cancer patient INK4B Gene mRNA expression level or p15 INK4B Step 1 for measuring the expression level of a protein; the p15 INK4B Gene mRNA expression level or p15 INK4B A second step of comparing the expression level of the protein with a control sample; and the p15 INK4B Gene mRNA expression level or p15 INK4B The invention provides a method for providing information necessary for diagnosing senescent colorectal cancer tumor cells, including a third step in which it is determined that the colorectal cancer tumor cells have aged when the expression level of the protein is higher than that of the control sample.

[0011] Another objective of the present invention is p15 INK4BThe invention provides a composition for inducing aging of colorectal cancer tumor cells comprising an expression or activity promoter as an active ingredient.

[0012] Another objective of the present invention is a first step of contacting colon cancer cells with a test substance; and p15 in the colon cancer cells contacted with the test substance INK4B A second step of measuring the degree of protein expression or activity; and the above p15 compared with a control sample INK4B The present invention provides a screening method for colorectal cancer treatments comprising a third step of selecting test substances in which the degree of protein expression or activity is increased. means of solving the problem

[0013] To achieve the above objective, the present invention p15 INK4B A biomarker composition for diagnosing senescent tumor cells of colorectal cancer is provided, comprising a protein or a gene encoding the same.

[0014] In addition, the present invention p15 INK4B A composition for diagnosing senescent tumor cells of colorectal cancer is provided, comprising as an active ingredient a preparation capable of measuring the expression or activity level of a protein, or the expression level of a gene encoding said protein.

[0015] In addition, the present invention provides a kit for diagnosing senescent tumor cells of colorectal cancer comprising the above composition.

[0016] In addition, the present invention relates to p15 from a sample isolated from a colorectal cancer patient. INK4B Gene mRNA expression level or p15 INK4B Step 1 for measuring the expression level of a protein; the p15 INK4B Gene mRNA expression level or p15 INK4B A second step of comparing the expression level of the protein with a control sample; and the p15 INK4B Gene mRNA expression level or p15 INK4BA method is provided for providing information necessary for diagnosing senescent colorectal cancer tumor cells, including a third step in which it is determined that the colorectal cancer tumor cells have aged when the expression level of the protein is higher than that of the control sample.

[0017] In addition, the present invention p15 INK4B A composition for inducing aging of colorectal cancer tumor cells is provided, comprising an expression or activity promoter as an active ingredient.

[0019] In addition, the present invention comprises a first step of contacting a test substance with colon cancer cells; and p15 in the colon cancer cells contacted with the test substance INK4B A second step of measuring the degree of protein expression or activity; and the above p15 compared with a control sample INK4B A screening method for colorectal cancer treatments is provided, comprising a third step of selecting test substances in which the degree of protein expression or activity is increased. Effects of the invention

[0020] The present invention is p15 INK4B The present invention relates to a biomarker composition for diagnosing senescent tumor cells of colorectal cancer comprising a protein or a gene encoding the same, wherein p15 is obtained through analysis after inducing senescence in cells extracted from colorectal cancer patients. INK4B this p16 INK4A Expression increases along with Confirmed its existence, and in cases where cellular senescence was also induced in vitro, p15 INK4B this p16 INK4A Confirmed that expression is increasing along with p15 INK4B의 The potential for diagnosing senescent tumor cells in colorectal cancer has been confirmed, and this can be utilized in various ways, such as biomarker compositions, diagnostic compositions, and diagnostic kits. Brief explanation of the drawing

[0021] Figure 1 shows the expression of p21, p27, and p53 and p16 in colorectal cancer cells. INK4A This is the result of confirming the correlation with. Figure 2 shows p14 in colon cancer cells ARF , H3K9me3 expression status and p16 INK4A This is the result of confirming the correlation with. Figure 3 is p16 INK4A - CDKN2B (p15 in positive cells INK4B This is the result of confirming whether the gene of ) is expressed. Figure 4 is p16 INK4A and p15 INK4B This is the result confirming that there is a positive correlation. Figure 5 shows the results of observing SA-b-Gal and heterochromatin foci in cancer cells in which cellular senescence was induced by treatment with hydrogen peroxide and doxorubicin. Figure 6 shows the results of observing gene expression in a cell aging model. Specific details for implementing the invention

[0022] The present invention will be described in more detail below.

[0024] The present invention is p15 INK4B A biomarker composition for diagnosing senescent tumor cells of colorectal cancer is provided, comprising a protein or a gene encoding the same.

[0025] The above biomarker composition for diagnosing senescent tumor cells in colorectal cancer is p16 INK4A It may additionally include proteins or genes encoding them.

[0026] The “diagnosis” of the present invention includes determining the susceptibility of an individual to a specific disease or condition, determining whether an individual currently has a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, or therametrics (e.g., monitoring the condition of an individual to provide information on therapeutic efficacy).

[0027] The "biomarker" of the present invention is a substance capable of diagnosing by distinguishing the tissue or cells of a subject with colorectal cancer senescent tumor cells from the tissue or cells of a normal control group, and includes organic biomolecules such as proteins or nucleic acids, lipids, glycolipids, glycoproteins, etc., which show an increasing or decreasing pattern in the tissue or cells of a subject with the disease compared to a normal control group.

[0029] In addition, the present invention p15 INK4B A composition for diagnosing senescent tumor cells of colorectal cancer is provided, comprising as an active ingredient a preparation capable of measuring the expression or activity level of a protein, or the expression level of a gene encoding said protein.

[0030] The above preparation may be a primer or probe that specifically binds to the gene, an antibody, peptide, peptide mimetics, aptamer, or compound that specifically binds to the protein, but is not limited thereto.

[0031] The term “primer” as used in the present invention refers to a short gene sequence that serves as a starting point for DNA synthesis, and means an oligonucleotide synthesized for the purpose of use in diagnosis, DNA sequencing, etc. The primers can typically be synthesized and used with a length of 15 to 30 base pairs, but this may vary depending on the purpose of use, and can be modified by methylation, capping, etc., by known methods.

[0032] As used in the present invention, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA of several to hundreds of base pairs in length, produced through enzymatic chemical separation, purification, or synthesis processes. The presence or absence of mRNA can be confirmed by labeling with radioisotopes or enzymes, and the probe can be designed and modified using known methods.

[0033] The term “antibody” as used in the present invention is a term known in the art and refers to a specific immunoglobulin directed toward an antigenic site. The antibody in the present invention is p15 of the present invention. INK4B It refers to an antibody that specifically binds to [the target], and the antibody can be manufactured according to conventional methods in the relevant technical field. The form of the antibody includes polyclonal antibodies or monoclonal antibodies, and includes all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

[0034] The term “peptide” as used in this invention has the advantage of high binding affinity to target substances and does not undergo denaturation even during heat or chemical treatment. In addition, due to its small molecular size, it can be attached to other proteins to be used as a fusion protein. Specifically, since it can be attached to high-molecular-weight protein chains, it can be used as a diagnostic kit and a drug delivery material.

[0035] The term “aptamer” as used in the present invention refers to a type of polynucleotide composed of a special class of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and is capable of binding to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to antigenic substances just like antibodies, but because they are composed of polynucleotides that are more stable than proteins, have a simple structure, and are easy to synthesize, they can be used as a substitute for antibodies.

[0037] In addition, the present invention provides a kit for diagnosing senescent tumor cells of colorectal cancer comprising a composition.

[0038] The “kit” of the present invention may include an antibody that specifically binds to a biomarker component, a secondary antibody conjugate conjugated with a label that develops color upon reaction with a substrate, a color-developing substrate solution to react with the label, a washing solution, and an enzyme reaction stopping solution, and may be manufactured as a plurality of separate packages or compartments containing the reagent components used.

[0040] In addition, the present invention relates to p15 from a sample isolated from a colorectal cancer patient. INK4B Gene mRNA expression level or p15 INK4B Step 1 for measuring the expression level of a protein; the p15 INK4B Gene mRNA expression level or p15 INK4B A second step of comparing the expression level of the protein with a control sample; and the p15 INK4B Gene mRNA expression level or p15 INK4B A method is provided for providing information necessary for diagnosing senescent colorectal cancer tumor cells, including a third step in which it is determined that the colorectal cancer tumor cells have aged when the expression level of the protein is higher than that of the control sample.

[0042] In addition, the present invention p15 INK4B A composition for inducing aging of colorectal cancer tumor cells is provided, comprising an expression or activity promoter as an active ingredient.

[0044] In addition, the present invention comprises a first step of contacting a test substance with colon cancer cells; and p15 in the colon cancer cells contacted with the test substance INK4B A second step of measuring the degree of protein expression or activity; and the above p15 compared with a control sample INK4B A screening method for colorectal cancer treatments is provided, comprising a third step of selecting test substances in which the degree of protein expression or activity is increased.

[0045] p15 above INK4BThe expression level may be one or more selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting, and flow cytometry (FACS), but is not limited thereto.

[0047] Hereinafter, to aid in understanding the present invention, examples and the like will be described in detail. However, the following examples and the like are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0049] <Experimental Example 1> Collection of Colorectal Cancer Patient Samples

[0050] Colorectal cancer samples were obtained via surgery from patients who had provided written consent at Ajou University Hospital. The study was approved by the Institutional Review Board of Ajou University Hospital (AJOUIRB-OBS-2016-218) and conducted in accordance with the Declaration of Helsinki. Fresh tumor tissue was collected immediately after surgery by a skilled pathologist and separated into two parts. One part was used to generate frozen tissue using liquid nitrogen freezing, while the second part was processed for paraffin tissue generation. To minimize potential bias due to genetic background, only colorectal cancer patients without microsatellite instability were selected. Patients who had received chemotherapy or radiation therapy prior to surgery were excluded.

[0052] <Experimental Example 2> Immunochemical Staining

[0053] Review of colorectal cancer tissue sections was performed independently by experienced pathologists. Some of the cut margin sections from fresh frozen tissue were used for SA-β-Gal staining, and other margin sections from FFPE tissue were used for immunohistochemical analysis. Immunohistochemical analysis was performed on paraffin blocks using a Benchmark XT automated processor (Ventana Medical Systems Inc, Tucson, AZ).

[0055] <Experimental Example 3> Preparation of Lentivirus

[0056] Lentivirus particles were prepared by infecting HEK-293TN cells with a lentivirus expression vector containing BRAF V600E and a packaging plasmid (pGagpol, pVSV-G). After 48 hours, the supernatant was harvested to extract lentivirus particles.

[0058] <Experimental Example 4> Cell Culture and Induction of Cellular Senescence

[0059] Colorectal cancer cell line SW480 was purchased from ATCC. SW480 was cultured in RPMI medium with 10% fetal bovine serum and 1% antibiotics. To establish a hydrogen peroxide and doxorubicin-induced cell line senescence model, 60 mM hydrogen peroxide and 50 nM doxorubicin were treated for 48 hours each. Subsequently, the medium was replaced with fresh medium, and a 48-hour stabilization period was observed. DAPI staining was performed at a 1:10,000 ratio to identify heterochromatin centrosomes. Human dermal fibroblasts were harvested from skin tissue and subcultured 3–7 times in DMEM medium supplemented with 10% fetal bovine serum. Human colorectal epithelial cells were purchased from Science Cell Research Laboratory and were used to establish an oncogene-induced cellular senescence model. Colorectal epithelial cells and fibroblasts were infected with BRAF V600E lentivirus for 5 days.

[0061] <Experimental Example 5> SA-b-Gal Staining

[0062] Frozen tissue slides were fixed with 10% paraformaldehyde for 1 minute and treated with SA-b-Gal solution in a 37°C incubator for 12 hours. Cultured cells were fixed with 4% paraformaldehyde and then treated with SA-b-Gal solution. After washing the cells with phosphate buffer solution, positive cells were observed under a light microscope.

[0064] <Experiment Example 6> Real-time Polymerase Chain Reaction (RT-PCR)

[0065] 1 μg of cellular RNA was used to generate a complementary DNA (cDNA) library using the SuperScript IV First-strand synthesis system. Real-time PCR was performed using the CyberGreen enzyme-solution mix under the following conditions (50 repetitions of 5 min at 95°C, 15 sec at 95°C, and 1 min at 60°C).

[0067] <Experimental Example 7> Analysis of Publicly Available Single-Cell RNA Sequencing Dataset

[0068] Analysis was performed using the open-source single-cell RNA sequencing dataset for colorectal cancer (GSE16655). This dataset includes 12 patients, of whom cells extracted from 6 microsatellite-stable patients (patients 7, 8, 17, 20, 21, and 25) were used in the experiment. Cells with a characteristic gene count of 200 to 10,000 were included in the experiment, while cells with a cellular-to-mitochondrial DNA ratio of 50% or more were excluded. To determine the dimensionality for Principal Component Analysis (PCA), Jackstraw scores were calculated using the 'ScoreJackStraw' function from the Seurat package, and 20 dimensions were determined to be appropriate for further analysis (p < 0.001). Cells with EPCAM > 1 and CDX2 > 1 gene counts were designated as cancer cells, and cancer cells with CDKN2A > 0 were designated as CDKN2A-positive STCs. To exclude inter-patient variation and batch effects, cells from Patient #8, which showed significant CDKN2A expression, were analyzed in greater detail. Jackstraw scores were calculated to re-cluster the cells of Patient #8, and three dimensions were determined to be suitable for subsequent principal component analysis (p < 0.001). To identify markers for CDKN2A-positive cancer cells, the Wilcoxon rank sum test was used with the 'FindMarkers' function from the "Seurat" package. Gene Set Enrichment Analysis (GSEA) was performed using the "fgsea" package in R (v 4.0.3). Permutations were performed 1,000 times, and p-values ​​were calculated using GSEA statistics. The weighted Kolmogorov-Smirnov statistic was calculated as the cumulative sum of the ranked gene list. Benjamin Hochberg (BH) adjusted p-values ​​were also calculated, and values ​​< 0.01 were considered statistically significant.

[0070] <Example 1> Confirmation of marker expression in existing senescent tumor cells

[0071] Colorectal cancer tissue was divided in half; one piece was prepared as frozen tissue and the other as paraffin tissue for the experiment. The closest segments from each piece were SA-b-Gal and p16 INK4A They were stained with [these] respectively. In addition, other cellular senescence markers p21, p27, p53, and p14 ARF and H3K9me3 on p16 INK4A I dyed it together with.

[0072] As a result, according to Figure 1, SA-b-Gal staining and p16, as indicated by the black arrow INK4A It was observed that the stained regions largely coincided, and other aging markers, p21, p27, and p53, were p16 INK4A It was confirmed that it is not expressed in common with.

[0073] In addition, according to Fig. 2, another aging marker, p16 INK4A p14 expressed via alternative splicing in the CDKN2A gene together with ARF In addition, it was confirmed that expression did not coincide with p16. H3K9me3, another aging marker, is p16 INK4A It was confirmed that although there is a positive correlation, the degree is not significant.

[0075] <Example 2> p15, a marker of senescent tumor cells in single-cell RNA sequencing INK4B excavation

[0076] When cancer cells were extracted by performing single-cell RNA sequencing on cells extracted from colorectal cancer patients, three major clusters were identified.

[0077] According to Fig. 3, cluster 1 (light green) cancer cells CDKN2A (p16 INK4AIt was expressing ), and conversely, cluster 2 (orange) and cluster 3 (sky blue) were observed to be expressing the cancer proliferation marker (MKI67), and when this was confirmed in a dot plot, CDKN2A (p16 INK4A It was observed that cells expressing ) were concentrated in Cluster 1. When GSEA analysis was performed on the oxidative stress-induced cellular senescence gene set, p16 INK4A - CDKN2B (p15 in positive cells INK4B It was observed that the gene expression of ) was increased. When this was confirmed using a dot plot, CDKN2A(p16 INK4A Cells expressing ) are CDKN2A(p16 INK4A Together with ), CDKN2B(p15 INK4B It was observed that ) is being expressed together.

[0078] In addition, when verifying this in patient tissue, according to Figure 4, p16 INK4A together with p15 INK4B We were able to observe a positive correlation, p16 INK4A In patients where this does not appear, p15 INK4B It was confirmed that it was not expressed together.

[0080] <Example 3> p15 when inducing cellular aging INK4B Confirmation of expression

[0081] To confirm that cellular senescence in colorectal cancer cells was induced normally, SA-b-Gal and heterochromatin foci were observed in colorectal cancer cells in which cellular senescence was induced by treatment with hydrogen peroxide and doxorubicin.

[0082] As a result, according to Figure 5, it was confirmed that SA-b-Gal was increased in cancer cells in which cellular senescence was induced by treatment with hydrogen peroxide and doxorubicin, and by observing heterochromatin foci, another in vitro cellular senescence marker, it was confirmed that cellular senescence was induced normally in both models.

[0083] As a result of observing gene expression in two cellular aging models using real-time PCR, according to Figure 6, p16 INK4A together with p15 INK4B By confirming that the mRNA of p15 increased, p15 as a marker for senescent colorectal cancer tumor cells INK4B The possibility of was confirmed.

[0085] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0086] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 p15 INK4B A kit for diagnosing senescent tumor cells of colorectal cancer comprising a preparation capable of measuring the expression or activity level of a protein, or the expression level of a gene encoding said protein. Claim 2 In claim 1, the diagnostic kit for colorectal cancer senescent tumor cells is p16 INK4A A kit for diagnosing senescent tumor cells in colorectal cancer, characterized by additionally including a preparation capable of measuring the expression level of a protein or a gene encoding it. Claim 3 A kit for diagnosing senescent tumor cells of colorectal cancer, characterized in that, in claim 1, the preparation is a primer or probe that specifically binds to the gene, an antibody, peptide, peptide mimetics, aptamer, or compound that specifically binds to the protein.

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