Dual-specificity phosphatase 23 as diagnostic marker for intrahepatic cholangiocarcinoma, and uses thereof

Abstract

The present invention relates to dual-specificity phosphatase 23 (DUSP23) as a diagnostic marker for intrahepatic cholangiocarcinoma and uses thereof. The present invention provides a use of DUSP23, which is a dual-specificity phosphatase, as a novel diagnostic marker for cholangiocarcinoma, including uses for diagnosis or prognosis. In addition, the present invention confirms that inhibition of DUSP23 expression induces a therapeutic effect on cholangiocarcinoma, and that such an effect is attributed to inhibition of YAP protein expression and suppression of YAP phosphorylation. Accordingly, DUSP23 can be advantageously applied for diagnosis, prognosis, or treatment of cholangiocarcinoma.

Description

Dual-Specific Phosphate 23 as a Diagnostic Marker for Intrahepatic Cholangiocarcinoma and Uses thereof

[0001] The present invention relates to a dual-specificity phosphatase 23 as a diagnostic marker for intrahepatic cholangiocarcinoma and its use.

[0002] The present application claims priority based on Korean Patent Applications No. 10-2024-0133019 and No. 10-2025-0136628, filed on September 30, 2024, and September 22, 2025, respectively, and all contents disclosed in the specifications and drawings of said applications are incorporated by reference into the present application.

[0003] Cholangiocarcinoma is a malignant tumor arising from the epithelial cells of the bile ducts. Depending on the location of the duct where it originates, it is classified into intrahepatic and extrahepatic cholangiocarcinoma, and the term is used to encompass gallbladder cancer or gallbladder cancer. Histologically, the majority of cholangiocarcinomas are adenocarcinomas, and the exact cause of the disease has not yet been clearly identified.

[0004] There are cases where cholangiocarcinoma is diagnosed after undergoing a cholecystectomy due to suspected gallstones caused by nonspecific symptoms or abnormal liver function tests; recently, with the widespread availability of health checkups, it is frequently diagnosed incidentally through abdominal ultrasound examinations. Furthermore, because the bile ducts are closely connected to vital organs such as the liver and pancreas, cancer metastasis to these organs is a factor that worsens the prognosis.

[0005] Currently, it is known that early diagnosis of cholangiocarcinoma is difficult because there are no symptoms in the early stages. Diagnosis of cholangiocarcinoma is performed using a combination of advanced imaging-based diagnostic methods, such as abdominal computed tomography, magnetic resonance imaging, and cholangioscopy; methods that test for tumor markers in the blood, such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and CA 125; and molecular biochemical tests that evaluate elevated levels due to liver dysfunction, such as alkaline phosphatase and gamma-glutamyl transpeptidase.

[0006] However, there are limitations in that accuracy is reduced due to the respective drawbacks. Image-based diagnosis makes it difficult to distinguish between extensive tumors and non-tumor conditions that may be masquerading as cholangiocarcinoma, and the accuracy is low because the blood markers used in molecular biochemical tests are not specific to cholangiocarcinoma and are indicators that can also appear in other solid tumors or infectious diseases.

[0007] In particular, since cholangiocarcinoma is similar to liver cancer and it is extremely difficult to distinguish between the two, there is a demand for diagnostic markers; however, no useful markers have been reported to date.

[0008] The object of the present invention is to provide a method for providing information for the diagnosis or prognosis prediction of bile duct cancer, comprising the following steps:

[0009] (S1) A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject; and

[0010] (S2) A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the expression level in a biological sample isolated from a control group.

[0011] Another objective of the present invention is to provide a composition for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising as an active ingredient a preparation for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

[0012] Another objective of the present invention is to provide a kit for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising the above-mentioned composition for diagnosing or predicting the prognosis of cholangiocarcinoma and instructions.

[0013] Another object of the present invention is to provide a screening method for a preparation for the prevention or treatment of bile duct cancer, comprising the following steps:

[0014] a) a step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a cholangiocarcinoma model administered a candidate substance; and

[0015] b) A step of selecting the candidate substance as a preventive or therapeutic agent for cholangiocarcinoma if the protein or mRNA expression level of DUSP23 in the isolated biological sample is reduced.

[0016] Another objective of the present invention is to provide a composition for screening agents for the prevention or treatment of cholangiocarcinoma, comprising as an active ingredient a agent for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

[0017] Another objective of the present invention is to provide a kit for screening agents for the prevention or treatment of cholangiocarcinoma, comprising the above-mentioned composition for screening agents for the prevention or treatment of cholangiocarcinoma and instructions.

[0018] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cholangiocarcinoma comprising an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient.

[0019] Another objective of the present invention is to provide a kit for the prevention or treatment of cholangiocarcinoma, comprising the above-mentioned pharmaceutical composition for the prevention or treatment of cholangiocarcinoma and instructions.

[0020]

[0021] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0022] The present invention provides a method for providing information for the diagnosis or prognosis prediction of bile duct cancer, comprising the following steps:

[0023] (S1) A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject; and

[0024] (S2) A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the expression level in a biological sample isolated from a control group.

[0025] In one embodiment of the present invention, the information providing method is,

[0026] (S3) A step of determining that the subject has cholangiocarcinoma or has a poor prognosis if the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the subject is higher than the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the control group; may be further included, but is not limited thereto.

[0027] In one embodiment of the present invention, the cholangiocarcinoma may be an intrahepatic cholangiocarcinoma, but is not limited thereto.

[0028] In one embodiment of the present invention, the intrahepatic cholangiocarcinoma may include early intrahepatic cholangiocarcinoma, but is not limited thereto.

[0029] In one embodiment of the present invention, the biological sample may be any one selected from the group consisting of bile duct tissue, blood, serum, whole blood, plasma, urine, saliva, tissue, cell, trachea, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen, but is not limited thereto.

[0030] In one embodiment of the present invention, the control group may be a normal individual or a liver cancer individual, but is not limited thereto.

[0031] The present invention provides a composition for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising as an active ingredient a preparation for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

[0032] In one embodiment of the present invention, the formulation may be any one selected from the group consisting of a primer set, a probe, an antibody or an antigen-binding fragment thereof, a ligand, an aptamer, a peptide, a protein, a compound, an agonist, an antagonist, and a reporter vector, but is not limited thereto.

[0033] The present invention provides a kit for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising the above-mentioned composition for diagnosing or predicting the prognosis of cholangiocarcinoma and instructions.

[0034] In one embodiment of the present invention, the description may provide a method for providing information for the diagnosis or prognosis prediction of bile duct cancer, but is not limited thereto.

[0035] The present invention provides a screening method for a preparation for the prevention or treatment of bile duct cancer, comprising the following steps:

[0036] a) a step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a cholangiocarcinoma model administered a candidate substance; and

[0037] b) A step of selecting the candidate substance as a preventive or therapeutic agent for cholangiocarcinoma if the protein or mRNA expression level of DUSP23 in the isolated biological sample is reduced.

[0038] The present invention provides a composition for screening agents for the prevention or treatment of cholangiocarcinoma, comprising as an active ingredient a agent for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

[0039] The present invention provides a kit for screening agents for the prevention or treatment of cholangiocarcinoma, comprising the above-mentioned composition for screening agents for the prevention or treatment of cholangiocarcinoma and instructions.

[0040] In one embodiment of the present invention, the description may teach a screening method for a preparation for the prevention or treatment of bile duct cancer, but is not limited thereto.

[0041]

[0042] The present invention provides a pharmaceutical composition for the prevention or treatment of bile duct cancer comprising an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient.

[0043] In one embodiment of the present invention, the expression or activity inhibitor may be one or more selected from the group consisting of siRNA, miRNA, shRNA, dsRNA, ASO (antisense oligonucleotide), CRISPR / CAS, compounds, peptides, aptamers, polynucleotides, antibodies, protacs, small molecules, and recombinant vectors, but is not limited thereto.

[0044] In one embodiment of the present invention, the composition may be characterized by one or more selected from the group consisting of the following, but is not limited thereto:

[0045] Decrease the protein level of YAP (yes-associated protein 1);

[0046] Does not affect YAP mRNA levels;

[0047] Increases the phosphorylation of YAP;

[0048] Inhibiting the movement of YAP to the nucleus; and

[0049] Inhibits the proliferation of bile duct cancer cells.

[0050] The present invention provides a kit for the prevention or treatment of bile duct cancer, comprising the above-mentioned pharmaceutical composition for the prevention or treatment of bile duct cancer and instructions.

[0051]

[0052] In addition, a method for treating bile duct cancer is provided, comprising the following steps:

[0053] A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject;

[0054] A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the gene or protein expression level in a biological sample isolated from a control group.

[0055] A step of determining that the subject has cholangiocarcinoma or has a poor prognosis if the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the subject is increased compared to the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the control group; and

[0056] A step of administering a therapeutic substance to a subject who has the above-mentioned bile duct cancer or is judged to have a poor prognosis.

[0057]

[0058] In addition, the present invention provides a method for preventing or treating bile duct cancer, comprising the step of administering a pharmaceutically effective amount of a composition containing an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient to an individual in need thereof.

[0059] In addition, the present invention provides a composition comprising an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient for the prevention or treatment of cholangiocarcinoma.

[0060] In addition, the present invention provides a use for manufacturing a preparation for the prevention or treatment of cholangiocarcinoma of a composition comprising an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient.

[0061]

[0062] In addition, the present invention provides a screening use for a composition comprising, as an active ingredient, a preparation for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) or the same, for the prevention or treatment of cholangiocarcinoma.

[0063] In addition, the present invention provides a use of a composition comprising, as an active ingredient, a protein of DUSP23 (Dual Specificity Phosphatase 23) or a preparation for measuring the mRNA level thereof, for manufacturing a preparation for screening of a preparation for the prevention or treatment of bile duct cancer.

[0064] According to the invention of Dual-specificity phosphatase 23 (DUSP23) as a diagnostic marker for intrahepatic cholangiocarcinoma and its uses, the invention provides a new diagnostic marker for cholangiocarcinoma, specifically the use of DUSP23, a dual-specificity phosphatase, for diagnosis or prognosis prediction. Furthermore, the invention confirms that a therapeutic effect on cholangiocarcinoma occurs when DUSP23 expression is inhibited, and confirms that this is due to the inhibition of YAP protein expression and the inhibition of YAP phosphorylation. Thus, it can be usefully utilized for the diagnosis, prognosis prediction, or treatment of cholangiocarcinoma.

[0065] Figures 1a to 1c show the experimental results confirming the use of DUSP23 for the diagnosis of cholangiocarcinoma. Specifically, Figure 1a confirms that the DUSP23 mRNA expression level is increased in cholangiocarcinoma through analysis of The Cancer Genome Atlas database. Figures 1b and 1c confirm the difference in the expression levels of the DUSP23 protein in normal liver tissue and cholangiocarcinoma tissue, respectively, through analysis of the Human Protein Atlas database.

[0066] Figure 1d shows the experimental results confirming the use of DUSP23 for predicting the prognosis of cholangiocarcinoma genes. Specifically, it was confirmed that the survival rate of patients decreased due to an increase in the expression level of DUSP23 mRNA.

[0067] Figures 2a and 2b show the experimental results confirming the use of the DUSP23 protein for the diagnosis of cholangiocarcinoma. Specifically, through immunohistochemistry, it was confirmed via the H-score that the expression of the DUSP23 protein is increased in the bile ducts of actual patients compared to normal bile ducts.

[0068] Figures 3a and 3b show the experimental results confirming the use of the DUSP23 protein for diagnosing cholangiocarcinoma, indicating that differential diagnosis is possible from liver cancer subjects. Specifically, it was confirmed through imaging analysis and H-score analysis, respectively, that there is a significant difference in the expression of the DUSP23 protein in liver cancer and cholangiocarcinoma cells.

[0069] Figure 3c shows the experimental results confirming the use of the DUSP23 gene for diagnosing cholangiocarcinoma, indicating that, similar to the DUSP23 protein, it is possible to differentiate diagnosis from liver cancer individuals. Specifically, it was confirmed that the expression levels of DUSP23 mRNA showed a significant difference between liver cancer and early cholangiocarcinoma cells.

[0070] Figure 4 shows that the proliferation of cancer cells is inhibited in cholangiocarcinoma cell lines with inhibited DUSP23 expression.

[0071] Figures 5a to 5c show that in DUSP23-inhibited cholangiocarcinoma cell lines, there is no change in the mRNA level of YAP, but the phosphorylation of YAP increases, the protein level of YAP decreases, and its translocation to the nucleus is inhibited.

[0072] Although bile duct cancer can be cured with surgery in the early stages, it is often asymptomatic until obstructive jaundice becomes clearly apparent; consequently, it is frequently diagnosed at an advanced stage, leading to a high number of missed opportunities for surgery.

[0073] For resectable cholangiocarcinoma, the 3-year survival rate is 45-60% and the average survival time is 18-30 months, while for cases where surgical resection is not possible, the survival rate is about 7 months. Since the survival rate is very low, diagnosing cholangiocarcinoma and presenting new treatment strategies are very important.

[0074] Currently, computed tomography (CT) is primarily used as an initial screening method for diagnosing cholangiocarcinoma; however, imaging diagnosis of cholangiocarcinoma often makes it difficult to differentiate it from primary hepatocellular carcinoma and metastatic liver cancer.

[0075] Although serum tumor markers (CA19-9 or CEA) are used in blood tests to diagnose cholangiocarcinoma, they have limitations as standalone diagnostic methods due to their low sensitivity and specificity, making them unsuitable for diagnosis or screening. Therefore, there is a need for diagnostic markers for cholangiocarcinoma with high sensitivity and specificity.

[0076] In order to solve such technical problems, the inventors of the present invention conducted a study and confirmed that the gene or protein expression of DUSP23 in the bile duct tissue of patients with cholangiocarcinoma increased compared to normal tissue. In addition, they confirmed the effect of inhibiting cancer cell proliferation through the inhibition of DUSP23 expression in the cholangiocarcinoma cell line SNU-1079, and confirmed that when DUSP23 expression was inhibited in the cholangiocarcinoma cell line SNU-1079, the phosphorylation of YAP increased and the expression of YAP decreased.

[0077] Accordingly, the present invention provides a method for providing information for the diagnosis or prognosis prediction of bile duct cancer, comprising the following steps:

[0078] (S1) A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject; and

[0079] (S2) A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the expression level in a biological sample isolated from a control group.

[0080] In one embodiment of the present invention, the information providing method is,

[0081] (S3) A step of determining that the subject has cholangiocarcinoma or has a poor prognosis if the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the subject is higher than the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the control group; may be further included, but is not limited thereto.

[0082] Accordingly, the present invention provides a method for providing information for the diagnosis or prognosis prediction of bile duct cancer, comprising the following steps:

[0083] (S1) A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject;

[0084] (S2) A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the expression level in a biological sample isolated from a control group; and

[0085] (S3) A step in which, if the level of protein or mRNA expression of DUSP23 in a biological sample isolated from a subject is increased compared to the level of protein or mRNA expression of DUSP23 in a biological sample isolated from a control, the subject is determined to have cholangiocarcinoma or have a poor prognosis.

[0086] In all claims below, the dual-specificity protein phosphatase 23, “DUSP23 (Dual Specificity Phosphatase 23),” is also known as low molecular mass dual-specificity phosphatase 3 (LDP-3) and is an enzyme encoded by the DUSP23 gene in humans. DUSP23 is not limited by a specific amino acid sequence or a base sequence encoding it, but may include any substance known as a dual-specificity dephosphatase 23, which is a protein represented by a sequence generally known in the art.

[0087] In this specification, including all claims below, “cholangiocarcinoma” may be defined as cancer that arises in the bile ducts. The bile ducts are tubes that carry bile produced in the liver to the duodenum. Bile is produced in hepatocytes, exits the liver, and is discharged through the duodenal papilla. The bile ducts are divided into intrahepatic bile ducts, which pass through the liver, and extrahepatic bile ducts, which extend beyond the liver to the duodenum. Cancers arising in these bile ducts can be classified according to their anatomical location into intrahepatic cholangiocarcinoma (20–25%), hilar cholangiocarcinoma (50–60%), and distal cholangiocarcinoma (20–25%).

[0088] Intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) are both cancers that originate in the bile ducts, but they differ significantly in their location of occurrence, clinical characteristics, and diagnostic and therapeutic approaches. Intrahepatic cholangiocarcinoma originates in the intrahepatic bile ducts within the liver parenchyma and is often confused with hepatocellular carcinoma (HCC) both imagingly and clinically. On the other hand, extrahepatic bile ducts (ECC) originate in the hilar region (including Klatskin tumors) or extrahepatic bile ducts (such as the common hepatic duct and common bile duct). Extrahepatic cholangiocarcinoma is often diagnosed relatively quickly due to the early onset of jaundice. In one embodiment of the present invention, the bile duct cancer may be intrahepatic cholangiocarcinoma, but is not limited thereto.

[0089] Meanwhile, early-stage intrahepatic cholangiocarcinoma can be significantly similar to hepatocellular carcinoma in clinical symptoms, blood test results, and imaging findings, which is a major factor making differential diagnosis difficult. For a definitive diagnosis, a comprehensive analysis of imaging characteristics, multiple evaluations of tumor markers, and biopsy and immunohistochemical detection must be performed in parallel.

[0090] However, in one embodiment of the present invention, it was confirmed that the protein expression level of DUSP23 in the cholangiocarcinoma tissue of a patient with early intrahepatic cholangiocarcinoma is significantly higher than that of a normal individual, thereby proving that early intrahepatic cholangiocarcinoma can be diagnosed solely by the protein expression level of DUSP23. Therefore, in one embodiment of the present invention, the intrahepatic cholangiocarcinoma may include early intrahepatic cholangiocarcinoma, but is not limited thereto. Accordingly, in one embodiment of the present invention, the cholangiocarcinoma is intrahepatic cholangiocarcinoma, and the intrahepatic cholangiocarcinoma may include early intrahepatic cholangiocarcinoma, but is not limited thereto.

[0091] In this case, early intrahepatic cholangiocarcinoma encompasses cases of early or stage 1 intrahepatic cholangiocarcinoma as generally defined in the art. Accordingly, in this specification, including the entire claims below, for example, early intrahepatic cholangiocarcinoma is defined as an early stage in which the cancer is confined only to the bile duct mucosa and muscle layer, where the cancer has not spread beyond the bile duct to surrounding organs or metastasized to lymph nodes, and may be defined as a state where the extent of tumor invasion is narrow. Alternatively, although the AJCC / UICC TNM staging is the de facto international standard and stage 1 of iCCA is defined as a single tumor without vascular invasion (≤5 cm: Stage IA, >5 cm: Stage IB), it is not limited thereto. Additionally, as per the entire claim below, early intrahepatic cholangiocarcinoma may mean Stage I (T1N0M0) of the AJCC / UICC TNM, and furthermore, as per the entire claim below, Stages I-II may be collectively referred to as “early” as a resectable (“resectable”) category in a clinical or surgical context, but are not limited thereto.

[0092] Furthermore, the present invention was completed by confirming that DUSP23 is significantly increased in bile duct tissue compared to liver cancer cells. Specifically, in one embodiment of the present invention, it was confirmed that the expression level of DUSP23 in the bile duct tissue of a bile duct cancer individual was significantly increased when compared not only to normal bile duct tissue but also to the bile duct of a liver cancer individual. Therefore, in one embodiment of the present invention, the control group may be a normal individual or a liver cancer individual, but is not limited thereto.

[0093] In one embodiment of the present invention, the biological sample may be any one selected from the group consisting of bile duct tissue, blood, serum, whole blood, plasma, urine, saliva, tissue, cell, trachea, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen, but is not limited thereto.

[0094] In this case, as per the entire claim below, the term "bile duct tissue" may include not only the bile duct tissue itself, but also cellular levels such as bile duct cells, normal cells, and cancer cells contained in the bile duct tissue.

[0095] The biological sample may be pretreated before use for detection or diagnosis. For example, this may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. The sample may be prepared to increase the detection sensitivity of protein markers, for example, the sample obtained from a subject may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction, or gel electrophoresis.

[0096] In one embodiment of the present invention, it was confirmed by methods such as mRNA level analysis, immunohistochemistry (IHC), or Western blot that both the protein of DUSP23 and the mRNA expression level thereof can be used as markers for the diagnosis or prognosis prediction of intrahepatic cholangiocarcinoma. Therefore, in this specification, including the entire claim below, the level of DUSP23 refers to both the protein and the mRNA expression level thereof. That is, since both the protein of DUSP23 and the mRNA expression level thereof can be used as the method for providing information for the diagnosis or prognosis prediction of cholangiocarcinoma according to the present invention, they can be utilized as markers that are simple yet exhibit high accuracy.

[0097] Including all claims below, the method for measuring protein levels is not limited to any specific method of measuring protein known in the art, provided it is by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted LaSer Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced LaSer Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, ELISA (enzyme linked immunosorbent assay), FACS, etc.

[0098] In all claims below, the method for measuring mRNA levels is not limited to any specific mRNA measurement method known in the art, provided it is by PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chip, and / or RNA chip.

[0099] In the entirety of the following claims, “the level is increased” means that something that was not previously detected is detected, or that the amount detected is greater than the normal level. In the entirety of the following claims, for example, “the level is increased” means that the level of the experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or higher, for example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or higher, and / or 0.5 times, 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or higher. In the entire specification including the following claims, specifically, it may mean an increase of 1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, or 10 times or more compared to that of the control group, but is not limited thereto. A person skilled in the art can understand the meaning of the opposite term as having the opposite meaning in accordance with the above definition.

[0100] In addition, in one embodiment of the present invention, the use of DUSP23 for prognostic prediction was verified by analyzing the overall survival rate and disease-free survival rate when applied to tissues of patients with cholangiocarcinoma. At this time, when the gene expression level of DUSP23 was measured and classified based on the median value, it was found that the prognosis of cholangiocarcinoma was poor in the patient group exhibiting DUSP23 gene expression levels above the median value. Therefore, in the present specification, including the full claims below, when DUSP23 is used for prognostic prediction, the expression level of DUSP23 may refer to the expression level of the gene, that is, mRNA. Furthermore, accordingly, in the present specification, including the full claims below, it may be extended to the expression level of highly expressed proteins. In the present invention, when analyzing survival rates, when dividing all patients into two groups—those with high expression and those with low expression—the variation in gene expression levels among patients is very large; therefore, if patients are divided based on the average value of gene expression levels, it becomes difficult to distinguish between patient groups. Thus, the overall survival rate and disease-free survival rate were analyzed by classifying patient groups based on the median value.

[0101] Accordingly, in all claims below, “poor prognosis” may mean a decrease in overall survival and disease-free survival, but is not limited thereto.

[0102] In this specification, including all claims below, the term “method for providing information” refers to a method for providing information regarding the diagnosis of a disease, which involves obtaining information regarding the onset or likelihood (risk) of a disease by analyzing biological samples of an individual or by confirming the increase or decrease in the expression level of the biomarker of the present invention. In this specification, including all claims below, for example, a method may be included for providing information regarding whether there is a possibility of cholangiocarcinoma developing in an individual, whether the likelihood of said cancer developing is relatively high, or whether said cancer has already developed, by measuring the level of the biomarker according to the present invention and comparing it with a control group. Furthermore, through a method using the biomarker of the present invention, it is possible to predict the risk of exacerbation due to the onset of the disease of the present invention, that is, whether the prognosis will be poor, and this may also be used as a method for providing information regarding the prevention and treatment of the disease of the present invention.

[0103] In this specification, including all claims below, the term “measurement” includes both detecting and confirming the presence (expression) of a target substance and detecting and confirming a change in the level of presence (expression level) of the target substance. In this case, measurement may be performed without limitation by including both qualitative methods (analysis) and quantitative methods. The types of qualitative and quantitative methods for measuring the presence of a substance according to the present invention are well known in the art, and the experimental methods described in this specification are included therein.

[0104] In the entirety of the claims below, the term “analysis” may preferably include “measurement,” wherein the qualitative analysis may mean measuring and confirming the presence of a target substance, and the quantitative analysis may mean measuring and confirming a change in the presence level (expression level) or amount of the target substance. In the present invention, analysis or measurement may be performed without limitation by including both qualitative and quantitative methods, and preferably, quantitative measurement may be performed.

[0105] In the entirety of the following claims, “confirmation” may have a broad sense including “measurement” and “analysis.”

[0106] In the entire specification including the following claims, “prediction” may include determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, and determining the prognosis of an object afflicted with a specific disease or condition (e.g., in the present invention, the subject of diagnosis is whether the disease or condition of the present invention occurs, and if prognosis prediction is included, it may include a prognosis regarding survival rate due to progression or deterioration of the disease or condition of the present invention, in which case a poor prognosis may be death, and in the case of cancer, it may include whether there is progression to metastasis). Or, in the entire specification including the following claims, it may have a broad meaning including all of the therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0107] In the entirety of the following claims, “diagnosis” includes determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object with a specific disease or condition (e.g., identification of tumor status, determination of tumor stage or determination of responsiveness of cancer to treatment, particularly in the present invention, diagnosis of cholangiocarcinoma or progression of the disease), or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0108] In the entirety of the following claims, the term “prognosis prediction” may mean predicting the degree of disease progression in a patient group of the disease of the present invention. For example, in the entirety of the following claims, it may mean predicting the probability of progression, deterioration, recurrence, maintenance, etc. of the disease condition through an increase or decrease in the level of the biomarker of the present invention.

[0109] In the entire specification including the following claims, the term “biomarker” means a marker that can distinguish between normal and pathological states or predict a therapeutic response and is objectively measurable. It has been confirmed that the level of DUSP23, a biomarker in biological samples of individuals with the disease of the present invention, differs from the increase or decrease in the respective levels of normal individuals or individuals with liver cancer, and thus it has been proven that the biomarker of the present invention can be used as a biomarker for the diagnosis or prognosis prediction of the disease of the present invention.

[0110] In addition, the present invention may provide a disease diagnostic device of the present invention for an individual. Specifically, the cancer may be cholangiocarcinoma, particularly intrahepatic cholangiocarcinoma. In the entirety of the following claims, the measuring unit of the diagnostic device may measure the expression level of a protein or gene using a preparation that measures the mRNA or protein expression level of DUSP23, a biomarker according to the present invention, with respect to a biological sample obtained from a subject. In the entirety of the following claims, by confirming the degree of expression of the protein or gene using the preparation in the measuring unit, the disease of the present invention may be diagnosed, or a high risk of onset may be diagnosed.

[0111] Including all claims below, the diagnostic device may further include a detection unit that predicts and outputs the presence or absence, stage, or type of the disease of the present invention of a subject from the degree of expression of the protein or gene obtained from a measurement unit.

[0112] Including all claims below, the detection unit can diagnose the disease of the present invention by generating and classifying information regarding the disease of the present invention according to the range of the expression level of the protein or gene obtained from the measurement unit.

[0113] In one embodiment of the present invention, it was confirmed that the survival rate of individuals with high gene expression levels of DUSP23 in bile duct cancer tissue was significantly reduced, and thus DUSP23, a biomarker of the present invention, can be used as a marker for predicting the prognosis of bile duct cancer.

[0114] The present invention provides a composition for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising as an active ingredient a preparation for measuring the gene or protein expression level of DUSP23 (Dual Specificity Phosphatase 23).

[0115] In one embodiment of the present invention, the cholangiocarcinoma may be an intrahepatic cholangiocarcinoma, but is not limited thereto.

[0116] In one embodiment of the present invention, the intrahepatic cholangiocarcinoma may include early intrahepatic cholangiocarcinoma, but is not limited thereto.

[0117] In one embodiment of the present invention, the formulation may be any one selected from the group consisting of a primer set, a probe, an antibody or an antigen-binding fragment thereof, a ligand, an aptamer, a peptide, a protein, a compound, an agonist, an antagonist, and a reporter vector, but is not limited thereto.

[0118] Including all claims below, the preparation capable of measuring the protein level may be one or more selected from the group consisting of antibodies, peptides, aptamers, proteins, and compounds that specifically bind to the protein, but is not limited thereto.

[0119] In the entirety of the following claims, the agent for measuring the miRNA level may be a primer, probe, oligonucleotide, antibody or antigen-binding fragment thereof that specifically binds to mRNA, a ligand, aptamer, peptide, agonist or antagonist, or a combination thereof, but is not limited thereto, and may mean any agent commonly used in the art.

[0120] Including all claims below, the “primer set” in this specification may be used to specifically amplify the mRNA of a corresponding marker to indirectly confirm the expression of the protein marker. In the present invention, the expression level of the marker can be quantitatively evaluated by performing reverse transcription polymerase chain reaction (RT-PCR) or quantitative PCR (qPCR) using the primer set. Therefore, the primer set can be usefully utilized for rapid and accurate monitoring of marker expression.

[0121] In the entirety of the following claims, a “probe” may serve to detect the presence of a target sequence by binding complementarily to a nucleic acid sequence derived from a protein marker. In the entirety of the following claims, for example, the expression of a marker may be confirmed by the intensity of a hybridization signal using a fluorescent or enzyme-labeled probe. Accordingly, the probe may be used as a key element to increase the sensitivity and specificity of marker detection.

[0122] In all claims below, as per this specification, an “antibody” can specifically bind to a protein marker to enable direct detection at the protein level. Antibodies can be applied to various analytical methods based on their high specificity and binding affinity. Therefore, antibodies can be utilized as an important component for the reliable detection and diagnosis of protein markers. In this case, as per all claims below, the presence and relative expression levels of a marker can be confirmed through immunoprecipitation, immunostaining, or immunoblotting methods using antibodies, but are not limited thereto, and the presence of expression, etc., can be analyzed through any method generally applied in the art.

[0123] In all claims below, the term “ligand” is a molecule capable of selectively binding to a specific receptor or protein structure to induce or inhibit a functional response, and such ligand may be used to detect the functional state of a biomarker based on whether the target molecule is activated or its binding characteristics. Depending on its unique binding affinity and selectivity, the ligand is applied to diagnostic, monitoring, or screening platforms.

[0124] In all claims below, the term “aptamer” refers to an oligonucleotide or peptide sequence that specifically binds to a protein marker and has binding characteristics similar to those of an antibody. Aptamers have the advantages of being easy to chemically synthesize and having high stability, allowing for repeated use. Therefore, aptamers can be usefully applied to diagnostic compositions for the selective binding and detection of protein markers.

[0125] In all claims below, the term “peptide” refers to a short amino acid sequence that selectively binds to a specific protein structure and is used to detect the expression level or activity state of a target protein. It can be combined with various labeling molecules and utilized in ELISA, SPR, or biosensor-based diagnostics. Because it is easy to chemically synthesize and its structure can be freely modified, it is widely applied as a ligand to enhance diagnostic specificity.

[0126] In all claims below, the “diagnostic protein” comprises a biologically derived or recombinant protein capable of indirectly reflecting an expression state based on interactions with a target biomarker or metabolic responses. These are coupled to a biosensor, microarray, or reporter system and used for the quantification or detection of a biomarker. The protein can induce signal amplification based on enzymatic activity, thereby enabling sensitive detection.

[0127] Including all claims below, the “small molecule compounds” described herein can determine the function or expression status of a biomarker through selective binding or reaction with a specific protein or receptor. These are generally utilized for diagnostic purposes based on binding reactions in high-speed screening platforms. They are chemically stable and capable of various structural modifications, offering high flexibility as diagnostic probes.

[0128] In all claims below, the term “agonist” in this specification refers to a substance that induces a physiological response by activating a specific receptor or protein and may be used to confirm the reactivity or functional status of a biomarker. For diagnostic purposes, the presence of a target molecule or its expression status may be indirectly estimated or confirmed through the induction of a specific response. Agonists are often utilized as a mechanism to induce a response in reporter assays or cell-based platforms.

[0129] In all claims below, as provided in this specification, a “antagonist” may be used to evaluate the functional status or signal transduction of a biomarker by binding to a specific receptor or enzyme and inhibiting its activation. In biosensors or functional diagnostic systems, the presence or absence of a target molecule can be determined through the difference in response before and after antagonist treatment. Highly specific antagonists serve as advantageous tools for highly sensitive function-based diagnostics.

[0130] Including all claims below, the “reporter vector” may be configured to include an expression regulation region of a protein marker so that a reporter gene is expressed according to expression activity. Since the reporter vector can be introduced into target cells to provide a signal corresponding to changes in marker expression visually or quantitatively, the reporter vector can be usefully utilized in protein marker-related research and the construction of detection systems.

[0131] The present invention provides a kit for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising the above-mentioned composition for diagnosing or predicting the prognosis of cholangiocarcinoma and instructions.

[0132] In one embodiment of the present invention, the description may provide a method for providing information for the diagnosis or prognosis prediction of bile duct cancer, but is not limited thereto.

[0133] In this specification, including all claims below, the term “kit” refers to a tool that additionally includes a formulation or a substance for the function, storage, etc., of the kit claimed in the present invention, which enables the use of the kit claimed in the present invention. In addition to the above substances, the kit of the present invention may include other components, compositions, solutions, devices, etc., which are typically required for the storage and processing methods thereof. In this case, each component may be applied one or more times without limitation on the number of times, there is no restriction on the order in which each substance is applied, and the application of each substance may proceed simultaneously or sequentially.

[0134] Including all claims below, the kit may include a container; instructions; etc. The container may serve to package the material and may also serve to store and secure it. The material of the container may take the form, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be equipped with a cap that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means and may also be equipped with a stopper that allows access to the contents by a needle. The kit may include an outer package, and the outer package may include instructions regarding the use of the components.

[0135]

[0136] The present invention provides a screening method for a preparation for the prevention or treatment of bile duct cancer, comprising the following steps:

[0137] a) a step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a cholangiocarcinoma model administered a candidate substance; and

[0138] b) A step of selecting the candidate substance as a preventive or therapeutic agent for cholangiocarcinoma if the protein or mRNA expression level of DUSP23 in the isolated biological sample is reduced.

[0139] In one embodiment of the present invention, the cholangiocarcinoma may be an intrahepatic cholangiocarcinoma, but is not limited thereto.

[0140] In one embodiment of the present invention, the intrahepatic cholangiocarcinoma may include early intrahepatic cholangiocarcinoma, but is not limited thereto.

[0141] The present invention provides a composition for screening agents for the prevention or treatment of cholangiocarcinoma, comprising as an active ingredient a agent for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

[0142] Including all claims below, the term “screening” in this specification may mean selecting a substance having a specific desired property from a candidate group of various substances by a specific operation or evaluation method.

[0143] That is, for the purposes of the present invention, the screening method of the present invention may refer to a series of processes including the step of determining the efficacy of a drug candidate substance by the said method in order to identify a therapeutic agent that produces the best therapeutic effect on an individual with bile duct cancer, but is not limited thereto.

[0144] Including all claims below, the step of confirming the therapeutic response and effect may be repeated several times depending on the therapeutic candidate substance, and may additionally include steps used in the art as general screening methods, such as adding additional substances or steps to confirm the therapeutic response and effect, but is not limited thereto.

[0145] Including all claims below, the cholangiocarcinoma subject to screening in this specification may be intrahepatic cholangiocarcinoma, but is not limited thereto.

[0146] In the entire specification including the following claims, “candidate substance” means an unknown substance used in screening to measure the increase or decrease in expression of the marker of the present invention by administering it to a target disease model in the present invention, and may be one or more selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, stem cells, stem cell culture media, compounds, microbial culture media or extracts, natural products, and natural extracts, but is not limited thereto.

[0147] In the entirety of the following claims, the term “treatment” refers to any act that improves or beneficially alters the target disease and the associated metabolic abnormalities, and may use methods such as chemotherapy, surgical procedures, or biological therapies.

[0148] In this case, as per the entire claim below, if the mRNA or protein expression level of the substance is increased or decreased depending on the type of comparison group, e.g., normal control group, liver cancer control group, etc., it may be determined that the cholangiocarcinoma has been treated (or improved). As per the entire claim below, the phrase “level is increased” is as described above.

[0149] Including all claims below, in this specification, a treatment method commonly used for treating a target disease in the present invention may be used, a commonly used therapeutic drug may be administered, and a candidate substance disclosed in the present invention may be administered, but is not limited thereto.

[0150] The present invention provides a kit for screening agents for the prevention or treatment of cholangiocarcinoma, comprising the above-mentioned composition for screening agents for the prevention or treatment of cholangiocarcinoma and instructions.

[0151] In one embodiment of the present invention, the description may teach a screening method for a preparation for the prevention or treatment of bile duct cancer, but is not limited thereto.

[0152] In this specification, including all claims below, the term “kit for screening agents for the prevention or treatment of cholangiocarcinoma” refers to a tool that enables screening agents for the prevention or treatment of cholangiocarcinoma, comprising the composition for screening agents for the prevention or treatment of cholangiocarcinoma according to the present invention and instructions. Any other details may be applied to the general provisions regarding the “kit” described in the present invention.

[0153]

[0154] The present invention provides a pharmaceutical composition for the prevention or treatment of bile duct cancer comprising an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient.

[0155] In one embodiment of the present invention, the expression or activity inhibitor may be one or more selected from the group consisting of siRNA, miRNA, shRNA, dsRNA, ASO (antisense oligonucleotide), CRISPR / CAS, compounds, peptides, aptamers, polynucleotides, antibodies, protacs, small molecules, and recombinant vectors, but is not limited thereto.

[0156] In the entirety of the following claims, “siRNA (small interfering RNA)” can perform the function of targeting and suppressing the expression of specific mRNA in the form of double-stranded RNA, and effectively reduces the expression of target genes through an RNA interference (RNAi) mechanism. Therefore, siRNA can be usefully utilized for the regulation of gene function and the development of therapeutic agents.

[0157] Including all claims below, “miRNA (micro RNA)” refers to an endogenous non-coding single-stranded RNA that can inhibit the translation of mRNA or induce its degradation, and can indirectly regulate the expression of target proteins by utilizing miRNA. Accordingly, miRNA can be utilized for gene expression regulation and disease research.

[0158] In all claims below, “shRNA (short hairpin RNA)” refers to RNA having a hairpin structure that induces a sustained RNA interference effect within a cell. As shRNA is delivered via a plasmid or viral vector to suppress the expression of a target gene, shRNA is suitable for long-term and stable gene suppression studies.

[0159] In all claims below, “dsRNA (double-stranded RNA)” is RNA with a double-stranded structure that can inhibit gene expression by degrading target mRNA. Since dsRNA can act through a mechanism similar to natural antiviral responses, dsRNA can be utilized in specific gene inhibition studies and as a candidate for therapeutic agents.

[0160] Including all claims below, “Antisense Oligonucleotide (ASO)” can bind complementarily to target mRNA to inhibit post-transcriptional expression and can be designed in the form of short single-stranded DNA or RNA. Thus, ASO can be applied as a means of selectively regulating target gene expression.

[0161] Including all claims below, “CRISPR / CAS (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein)” corrects or inhibits gene sequences by cleaving target DNA. In the present invention, it has been confirmed that inhibition or removal of gene activity can be performed through a CRISPR / CAS system.

[0162] In all claims below, the term “chemical compound” refers to a molecule having an organic or inorganic chemical structure that inhibits specific enzymes or receptors to suppress the expression or activity of proteins. The compound may be prepared from natural or synthetic compounds and may be applied to inhibitors requiring rapid onset of efficacy.

[0163] In this specification, including all claims below, “peptide” refers to a short polypeptide composed of amino acids that can bind to a target protein and inhibit its function. Since peptides are easy to synthesize and have high target specificity, they can be utilized as various in vivo inhibitory compositions.

[0164] In all claims below, the “aptamer” is a single-stranded oligonucleotide or peptide capable of specifically binding to a protein or other molecule. Since the aptamer is easy to chemically synthesize and has high stability, allowing for repeated use, the aptamer can be utilized as a formulation that combines selective binding and inhibition functions.

[0165] In all claims below, the “polynucleotide” consists of a continuous sequence of nucleotides and can inhibit or regulate gene expression. Since the polynucleotide is inserted into a vector and exhibits an expression-inhibiting effect within a cell, it can be applied to various gene inhibition studies.

[0166] In all claims below, the “antibody” selectively binds to a target protein and blocks the activity of said protein. Since the antibody can be utilized for various biological inhibitions based on its high specificity and binding affinity, it can be used as a reliable biological agent for inhibition.

[0167] In all claims below, “PROTAC (proteolysis-targeting chimera)” is a dual-function compound that induces a target protein to degrade it via the ubiquitin-proteasome pathway. The PROTAC is composed of a linker that connects ligands and can be utilized as a selective protein removal agent by simultaneously binding a target protein and an E3 ligase.

[0168] In all claims below, the “small molecule” is an organic compound with a low molecular weight that inhibits the activity of an enzyme or receptor within a cell. Since the small molecule is easy to synthesize and has excellent drug delivery properties, the small molecule can be utilized as an inhibitor or therapeutic agent for various target proteins.

[0169] In all claims below, the “recombinant vector” regulates the expression of a desired protein within a cell by inserting a specific gene sequence. The recombinant vector may be produced in various forms, such as plasmids or viral vectors, and may be applied for research on gene expression inhibition and for therapeutic purposes.

[0170] In one embodiment of the present invention, it was confirmed that a therapeutic effect on intrahepatic cholangiocarcinoma was exhibited when DUSP23 expression was inhibited by treatment with shRNA oligonucleotide. Accordingly, it was proven that cholangiocarcinoma can be treated by treating with a substance that inhibits the expression or activity of DUSP23. shRNA oligonucleotide was used as an example of a preparation to inhibit DUSP23 expression or activity. Therefore, it may be obvious that the preventive or therapeutic effect on intrahepatic cholangiocarcinoma does not occur depending on the specific type of preparation of a substance that inhibits the expression or activity of DUSP23, such as a compound or siRNA, and in the present invention, a preparation for inhibiting expression or activity may refer to any substance that inhibits the expression or activity of the marker of the present invention.

[0171] In one embodiment of the present invention, in the cholangiocarcinoma cell line SNU-1079 in which DUSP23 expression is inhibited, it was found that while there is no difference in the mRNA expression level of YAP, the protein expression level of YAP decreases and the phosphorylation of YAP increases. Accordingly, it was also confirmed that the translocation of YAP to the nucleus is inhibited. It was confirmed that the therapeutic activity of the present invention for the prevention of cholangiocarcinoma, which occurs due to the inhibition of DUSP23 expression, arises through the inhibition of YAP expression at the protein level rather than the gene level, increased YAP phosphorylation, and inhibition of YAP translocation to the nucleus. Therefore, in one embodiment of the present invention, the pharmaceutical composition may additionally include a YAP protein expression or activity inhibitor, but is not limited thereto. In this case, as per the entire claim below, the term “expression or activity inhibitor” may be understood as a substitute for the aforementioned agent.

[0172] In all claims below, “YAP (yes-associated protein 1)”, also known as YAP1 or YAP65, is a protein that acts as a transcriptional co-regulator that promotes the transcription of genes involved in cell proliferation and inhibits apoptosis genes. YAP is a component of the Hippo signaling pathway that regulates organ size, regeneration, and tumor formation. YAP was first identified due to its ability to associate with the SH3 domains of Yes and Src protein tyrosine kinases and is known as a potent oncogene that is amplified in various human cancers.

[0173] Since YAP is a signaling pathway that plays a major role in the activity of intrahepatic cholangiocarcinoma, the present invention confirmed changes in the YAP gene or protein levels in intrahepatic cholangiocarcinoma cell lines in which DUSP23 expression is suppressed. As a result, in one embodiment of the present invention, the composition may be characterized by one or more selected from the group consisting of the following, but is not limited thereto:

[0174] Decrease the protein level of YAP (yes-associated protein 1);

[0175] No effect on YAP mRNA levels;

[0176] Increasing the phosphorylation of YAP; and

[0177] Hinders the movement of YAP to the nucleus.

[0178] In the entirety of the following claims, “pYAP(Ser127)” may refer to YAP in which a phosphate group is attached to serine, the 127th amino acid of YAP, in a phosphorylated form. It is known that phosphorylating Ser127 of YAP creates a binding site for 14-3-3, which allows YAP to be retained in the cytoplasm, but is not limited thereto. In the entirety of the following claims, the amino acid sequence of YAP may include any known sequence.

[0179] Meanwhile, phosphorylation of YAP can occur at various amino acids in addition to the 127th amino acid. For example, it is known that phosphorylating Ser381 of YAP leads to the recognition of YAP and its degradation by ubiquitination via SCF-β-TrCP.

[0180] In one embodiment of the present invention, the composition may inhibit the proliferation of bile duct cancer cells, but is not limited thereto.

[0181] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric granules, liquids, eye drops, ellipsoids, emulsions, suspensions, ethanol tablets, troches, fragrances, limonades, tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols, according to conventional methods, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms.

[0182] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0183] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0184] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to tablets, powders, granules, capsules, pills, and lozenges according to the present invention; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0185] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucroses, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0186] In the syrup preparation according to the present invention, a solution of white sugar, other sugars or sweeteners, etc. may be used, and if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc. may be used.

[0187] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0188] In the suspension agent according to the present invention, suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910 may be used, and surfactants, preservatives, stabilizers, coloring agents, and fragrances may be used as needed.

[0189] The injectable preparation according to the present invention comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-inflammatory agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0190] The suppository according to the present invention comprises cocoa dough, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, monollene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroccote SP, S-70-XXA, S-70-XX75 (S-70-XX95). Bases such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0191] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0192] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0193] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0194] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention belongs.

[0195] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0196] The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease.

[0197] In the entire specification including the following claims, the term “individual” means an object requiring disease risk prediction, diagnosis, prognosis prediction, or treatment, and more specifically may mean mammals such as human or non-human primates, mice, rats, dogs, cats, horses, and cattle, but is not limited thereto.

[0198] In all claims below, the term “determination” may be used interchangeably with “discrimination” or “determination” and means distinguishing an object according to specific criteria. In the present invention, it may be used to mean distinguishing whether an object is diagnosed as having or likely to have a molecular subtype or detailed subtype of thyroid cancer, distinguishing whether said object will exhibit a therapeutic effect for each specific treatment for thyroid cancer, or distinguishing whether said object has or does not have sensitivity to said combination of treatments, but is not limited thereto.

[0199] Including all claims below, the term “administration” in this specification means providing a specific composition of the present invention to an individual by any appropriate method.

[0200] The present invention provides a kit for the prevention or treatment of bile duct cancer, comprising the above-mentioned pharmaceutical composition for the prevention or treatment of bile duct cancer and instructions.

[0201] In one embodiment of the present invention, the description may teach one or more treatment methods provided in the present invention as follows, but is not limited thereto.

[0202] In addition, a method for treating bile duct cancer is provided, comprising the following steps:

[0203] (S1) A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject; and

[0204] (S2) A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the expression level in a biological sample isolated from a control group;

[0205] (S3) A step of determining that the subject has cholangiocarcinoma or has a poor prognosis if the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the subject is increased compared to the level of protein or mRNA expression of DUSP23 in a biological sample isolated from the control; and

[0206] (S4) A step of administering a therapeutic substance to a subject who has the above-mentioned bile duct cancer or is judged to have a poor prognosis.

[0207] Alternatively, the present invention provides a method for preventing or treating cholangiocarcinoma, comprising the step of administering a pharmaceutically effective amount of a composition containing an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient to an individual in need thereof.

[0208] Including all claims below, the therapeutic material treated in the treatment method of the present invention may include, but is not limited to, the therapeutic pharmaceutical composition of the present invention, and may also be a material generally treated in the art to treat cholangiocarcinoma.

[0209] In all claims below, the terms “pharmaceuticalally acceptable” or “pharmaceuticalally effective” mean a compound or composition that is suitable for use in contact with tissues of a subject (e.g., human) and is within the scope of sound medical judgment, having a reasonable benefit-risk ratio without excessive toxicity, irritation, allergic reaction, or other problems or complications.

[0210] Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Acid addition salts can be prepared by conventional methods, for example, by dissolving a compound in an excess amount of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, they can be prepared by heating an equal molar amount of the compound and an acid or alcohol in water, followed by drying the mixture by evaporation, or by suction filtration of the precipitated salt.

[0211] Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium, but are not limited thereto. Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. In this case, it is particularly suitable for pharmaceutical purposes to produce sodium, potassium, or calcium salts as metal salts, and the corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0212] In this specification, including all claims below, the term “kit” refers to a tool that enables the prevention or treatment of an individual determined to have the target disease of the present invention, comprising the pharmaceutical composition for the prevention or treatment of cholangiocarcinoma and instructions. Any other details may be applied to the general provisions regarding the “kit” described in the present invention.

[0213]

[0214] In all claims below, when the term “comprising” is used in this specification, it means that other components may be included, rather than excluding other components, unless specifically stated otherwise. In all claims below, the terms “a step” or “a step of” as used in this specification do not mean “a step for”.

[0215] The terms used in this specification, including all claims below, have been selected to be as widely used as possible while considering their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been selected at the applicant's discretion, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the present invention.

[0216] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0217]

[0218] [Example]

[0219]

[0220] immunohistochemistry

[0221] Tissue samples were fixed with 10% neutral buffered formalin (#010-1406-1010; GD CHEM, Chungcheongbuk-do, South Korea) and embedded in paraffin blocks. Paraffin-embedded tissues (FFPEs) were sectioned to a thickness of 4 μm. For staining, the sectioned slides were stored at 60°C for 12 hours, after which deparaffinization and hydration were performed by treating with xylene for 10 minutes, followed by sequential treatment with 100% ethanol for 10 minutes, 95% ethanol for 5 minutes, 70% ethanol for 5 minutes, 50% ethanol for 5 minutes, and PBS for 10 minutes. The samples were permeated with PBS containing 0.1% Triton X-100 for 10 minutes, followed by blocking with normal goat serum for 1 hour. Subsequently, the primary antibody targeting DUSP23 was treated at 4°C for 20 hours. To detect DUSP23, a secondary antibody targeting the primary antibody and VECTASTAIN were used for the staining process. 　 Elite 　 The procedure was performed according to the manufacturer's instructions using the ABC-HRP Kit, Peroxidase (Mouse IgG; #PK-6102) and the DAB Substrate Kit, Peroxidase (HRP), with Nickel, and (3,3'-diaminobenzidine; #SK-4100).

[0222]

[0223] Western blotting technique

[0224] Tissue samples were lysed with RIPA solution, and SNU-1079 cells were washed with PBS and lysed with a lysis buffer containing 1% NP-40, 1% sodium dodecyl sulfate (SDS), 150 mM sodium chloride (NaCl), 6 mM sodium hydrogen phosphate (Na2HPO4), 4 mM sodium dihydrogen phosphate (NaH2PO4), 2 mM EDTA, 50 mM sodium fluoride (NaF), 1 mM sodium orthovanadate (Na3VO4), 1 mM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride (PMSF). Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride membrane (PVDF membrane; #IPVH00010, Millipore), blocked with a 0.1% Triton X-100 PBS solution containing 5% skim milk, and incubated with a primary antibody. Subsequently, the samples were incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (Jackson ImmunoResearch Laboratories), and the protein detection signal was reacted using an enhanced chemiluminescence (ECL) reagent and detected using a Fusion Solo S imaging system (VILBER, Collegien, France).

[0225]

[0226] RNA extraction and real-time reverse transcriptase chain reaction

[0227] SNU-1079 cells were washed with PBS, and then lysed using RNAiso Plus reagent to isolate total RNA, following the manufacturer's instructions. PrimeScript reverse transcriptase and RNase inhibitor were used to synthesize complementary DNA from the isolated RNA, and SYBR Premix Ex Taq II reagent and QuantStudio 3 were used for real-time polymerase chain reaction according to the manufacturer's instructions. The primers used for the real-time polymerase chain reaction are as follows. For the YAP gene, forward (5'-GACTTCCTGAACAGTGTGGA-3'; seq. 2) and reverse (5'-ATGGCTTCAAGGTAGTCTGG-3'; seq. 3) primers were used, and for the GAPDH gene, forward (5'-GACCCCTTCATTGACCTC-3'; seq. 4) and reverse (5'-TCCTGGAAGATGGTGATG-3'; seq. 5) primers were used. Relative gene expression levels were measured using the 2-ΔΔCt quantification method.

[0228]

[0229] immunocytochemistry

[0230] Approximately 2 × 10^4 SNU-1079 cells were cultured for 48 hours on 12 mm coverslips coated with 50 μg / ml type I collagen and fixed by treatment with 3.7% paraformaldehyde. After permeation treatment with a PBS solution containing 0.5% Triton X-100 for 10 minutes, the cells were blocked by treatment with a 0.1% PBS-Triton X-100 solution containing 2% BSA for 1 hour. Next, a primary antibody targeting YAP was treated for 1 hour, followed by washing three times with a 0.1% PBS-Triton X-100 solution for 10 minutes, and the proteins were stained by treatment with a secondary antibody conjugated with a fluorescent dye for 1 hour. After mounting the stained samples onto a slide glass using Fluoromount-G (SouthernBiotech, AL, USA), images were captured using an Eclipse 80i fluorescence microscope (Nikon) and a digital camera DS-Qi2 (Nikon), and the images were analyzed using the microscope software NIS-Elements Advanced Research (Nikon).

[0231]

[0232] Example 1. Increased DUSP23 expression in intrahepatic cholangiocarcinoma patient tissues based on bioinformatics database analysis, and confirmation of its use for diagnosis or prognosis prediction

[0233] Example 1-1. Diagnostic use of DUSP23

[0234] The gene expression levels of DUSP23 in 9 healthy individuals and 35 patients with intrahepatic cholangiocarcinoma were analyzed using a web program (http: / gepia.cancer-pku.cn) capable of analyzing RNA sequencing data from The Cancer Genome Atlas (TCGA), a bioinformatics database, on tissues of patients with intrahepatic cholangiocarcinoma.

[0235]

[0236] As a result, it was confirmed that gene expression of DUSP23 was significantly increased in patients with intrahepatic cholangiocarcinoma (Fig. 1a).

[0237]

[0238] In addition, the expression levels of DUSP23 protein in normal and cholangiocarcinoma tissues were analyzed using the Human protein ATLAS database.

[0239]

[0240] As a result (Figs. 1b and 1c), it was confirmed that the expression levels of the DUSP23 protein differed between normal tissue and intrahepatic cholangiocarcinoma tissue. According to this, the expression level of the DUSP23 protein in cholangiocytes of normal tissue was found to be low, whereas a significant increase in the expression of DUSP23 was observed in intrahepatic cholangiocarcinoma tissue.

[0241]

[0242] In conclusion, it was proven that DUSP23 can be used as a diagnostic marker for intrahepatic cholangiocarcinoma, as it exhibits different expression levels in normal tissue and cholangiocarcinoma tissue at both the gene and protein levels.

[0243]

[0244] Examples 1-2. Use of DUSP23 for prognosis prediction

[0245] In this embodiment, the use of DUSP23 for predicting the prognosis of intrahepatic cholangiocarcinoma was investigated. Specifically, the overall survival rate and disease-free survival rate of patients with intrahepatic cholangiocarcinoma were analyzed according to the expression level of DUSP23 using the Kaplan-Meier assay.

[0246]

[0247] As a result, based on the DUSP23 gene expression levels of patients with cholangiocarcinoma, patients with expression above the median were classified into the high DUSP23 group (18 patients, indicated by the red line in Fig. 1d), and patients with expression below the median were classified into the low DUSP23 group (18 patients, indicated by the blue line in Fig. 1d). Analysis of the survival rates of the two groups revealed that overall survival (HR = 1.5) and disease-free survival decreased in the group of patients with high DUSP23 expression (HR = 2.9; Fig. 1d).

[0248]

[0249] According to these results, DUSP23 was confirmed to be a marker capable of not only diagnosing cholangiocarcinoma but also predicting its prognosis.

[0250]

[0251] Example 2. Increased DUSP23 Expression in Cholangiocarcinoma Patient Tissues by Immunohistochemical Analysis and Confirmation of Its Diagnostic Use

[0252] Based on the results of Example 1, an analysis was conducted to determine whether the expression of DUSP23 was increased in the tissues of actual cholangiocarcinoma patients. For this analysis, the histochemical score (H-score) was derived using the immunohistochemistry (IHC) technique, and the Western blot method was performed. DUSP23 protein staining was performed using the immunohistochemistry technique with DUSP23 antibodies on liver tissues from five normal individuals and cancer tissues from five intrahepatic cholangiocarcinoma patients.

[0253]

[0254] As a result (Fig. 2a), the level of DUSP23 protein expression can be confirmed by the degree of brown coloration, and it was confirmed that DUSP23 protein expression was significantly increased in the tissue of patients with intrahepatic cholangiocarcinoma compared to normal bile ducts.

[0255]

[0256] The expression levels of DUSP23 protein in biliary tissues from healthy individuals and tissues from patients with intrahepatic cholangiocarcinoma were compared by analyzing H-scores. Using the IHC Profiler Plugin function of the Image J web program, the level of DUSP23 protein expression was classified into four categories—negative, weak, moderate, and strong—through an automated digital IHC image analysis algorithm. The H-score was calculated by considering the area ratios occupied by each expression level. The following formula was used for the H-score: H-score = (0 X negative staining area ratio) + (1 X weak staining area ratio) + (2 X moderate staining area ratio) + (3 X strong staining area ratio). This calculation method was referenced from the paper *Varghese F, Bukhari AB, Malhotra R, De A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS One. (2014).

[0257]

[0258] As a result, it was confirmed that the expression of DUSP23 protein was increased in cholangiocarcinoma tissue (Fig. 2b, left graph). Subsequently, the expression levels of DUSP23 protein were determined using the Western blot method with four samples each of normal liver tissue and cholangiocarcinoma tissue, and it was confirmed that the expression of DUSP23 protein was increased, consistent with the results of the immunohistochemistry method (right figure of Fig. 2b).

[0259]

[0260] In conclusion, similar to the results of Example 1, when analyzed using immunohistochemistry, it was confirmed via H-score that the expression of the DUSP23 protein was increased in the bile ducts of actual cholangiocarcinoma patients compared to normal bile ducts. Since this increase in DUSP23 expression at the gene or protein level is statistically significant, it was re-proven that DUSP23 is a marker capable of distinguishing and diagnosing cholangiocarcinoma from normal individuals.

[0261]

[0262] Example 3. Confirmation of the diagnostic use of DUSP23 to distinguish intrahepatic cholangiocarcinoma, including early intrahepatic cholangiocarcinoma, from liver cancer

[0263] In Examples 1 and 2, it was confirmed that the gene or protein expression level of DUSP23 in the bile duct tissue of patients with cholangiocarcinoma was significantly increased compared to normal individuals. Meanwhile, since it is quite difficult to distinguish between liver cancer and intrahepatic cholangiocarcinoma using existing methods for diagnosing intrahepatic cholangiocarcinoma, we analyzed whether it is possible to diagnose intrahepatic cholangiocarcinoma, particularly early-stage intrahepatic cholangiocarcinoma, by distinguishing it from liver cancer using DUSP23.

[0264]

[0265] Example 3-1. Confirmation of the use of DUSP23 for comparative diagnosis of intrahepatic cholangiocarcinoma and liver cancer

[0266] First, we analyzed whether DUSP23 could be used to differentiate and diagnose intrahepatic cholangiocarcinoma from liver cancer. Specifically, we analyzed slides of liver tissue from five normal individuals, hepatocellular carcinoma (HCC) tissue from five individuals, and cancer tissue from five patients with intrahepatic cholangiocarcinoma (iCCA). In both the normal and cholangiocarcinoma samples, bile ducts were identified in all slides, and analysis was performed on all samples. Among the five HCC samples, bile ducts were identified in only two slides, and approximately 100 bile ducts were analyzed from these two slides. The analysis was performed using the image-J IHC profiler plug-in, in the same manner as in Example 2.

[0267]

[0268] As a result (Figs. 3a and 3b), the H-score for DUSP23 in the bile ducts of the cholangiocarcinoma sample (iCCA) was found to be significantly higher compared to the bile ducts of the normal sample, and this was confirmed to be statistically significant. In particular, the H-score for DUSP23 in the bile ducts of the cholangiocarcinoma sample (iCCA) was also found to be significantly higher compared to the bile ducts of the hepatocellular carcinoma sample (HCC), and this was found to be statistically significant.

[0269]

[0270] Example 3-2. Confirmation of the use of DUSP23 for comparative diagnosis of early cholangiocarcinoma and liver cancer

[0271] In Example 3-2, we analyzed whether the ability to distinguish and diagnose cholangiocarcinoma from liver cancer confirmed in Example 3-1 also applies to early-stage cholangiocarcinoma. Specifically, to compare expression in the early stages of cancer, we compared gene expression using only patient data from a specific stage, stage I.

[0272]

[0273] As a result (Fig. 3c), the expression level of the DUSP23 gene was observed to be higher in the tissue of patients with Stage I cholangiocarcinoma compared to normal liver tissue, and this was confirmed to be statistically significant.

[0274]

[0275] Overall, it was confirmed that the protein expression level of DUSP23 can distinguish cholangiocarcinoma from both normal individuals and liver cancer patients, and that the gene expression level is a marker that can distinguish from early-stage cholangiocarcinoma from normal individuals and liver cancer patients.

[0276]

[0277] Example 4. Confirmation of therapeutic activity for cholangiocarcinoma due to inhibition of DUSP23 expression

[0278] Based on the experimental results of Example 3, we analyzed whether therapeutic activity for cholangiocarcinoma occurs with the inhibition of DUSP23 expression. To confirm this, a cholangiocarcinoma cell line with inhibited DUSP23 expression was constructed, and the inhibition of proliferation of the cell line was verified. An shRNA lentivirus system targeting the coding region of the DUSP23 gene was used to construct the intrahepatic cholangiocarcinoma cell line with inhibited DUSP23 expression. The constructed shRNA oligonucleotide targeting DUSP23 (5'-GCTGAAATCCGACGACTACGA-3'; seq. 1.) was cloned into the pLKO.1-puro vector, and the lentivirus helper plasmids psPAX2 and pMD2.G were co-transformed into human embryonic kidney cell line 293T and cultured for 72 hours to obtain a DUSP23 shRNA-containing lentivirus produced in 293T. The acquired lentivirus was infected into the intrahepatic cholangiocarcinoma cell line SNU-1079 by treating it with 8 μg / ml polybrene to induce inhibition of DUSP23 gene expression, and inhibition of DUSP23 gene expression was confirmed by Western blot technique.

[0279]

[0280] As a result (Fig. 4), it was confirmed that the growth of cancer cells was inhibited in the cholangiocarcinoma cell line SNU-1079 in which DUSP23 expression was inhibited. These experimental results suggest that cholangiocarcinoma can be treated by treating with a substance that inhibits the expression or activity of DUSP23.

[0281]

[0282] Example 5. Confirmation of the mechanism of therapeutic activity for cholangiocarcinoma following inhibition of DUSP23 expression

[0283] In Example 4, it was shown that inhibiting the expression of DUSP23 inhibited the proliferation of cholangiocarcinoma cells. Meanwhile, the YAP (yes-associated protein 1) signaling pathway plays a major role in the activity of intrahepatic cholangiocarcinoma. Therefore, in Example 5, using the intrahepatic cholangiocarcinoma cell line with inhibited DUSP23 expression used in Example 4, the relationship between increased DUSP23 expression and YAP signaling activity in intrahepatic cholangiocarcinoma was verified. RNA extraction and real-time reverse transcription polymerase chain reaction were performed to confirm changes in YAP gene expression following the inhibition of DUSP23 expression, and Western blot was performed to confirm changes in the YAP signaling pathway.

[0284]

[0285] As a result, in the cholangiocarcinoma cell line SNU-1079 with inhibited DUSP23 expression, there was no difference in the mRNA expression level of YAP, but the protein expression level of YAP decreased and the phosphorylation of YAP increased. In addition, it was confirmed that the translocation of YAP to the nucleus was inhibited (Figs. 5a to 5c).

[0286]

[0287] According to these results, the therapeutic activity for cholangiocarcinoma resulting from the inhibition of DUSP23 expression was confirmed to occur through inhibition of YAP expression at the protein level rather than the gene level, increased YAP phosphorylation, and inhibition of YAP translocation to the nucleus.

[0288]

[0289] 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.

[0290] The present invention relates to a dual-specificity phosphatase 23 (DUSP23) as a diagnostic marker for intrahepatic cholangiocarcinoma and its uses. Specifically, the invention provides a dual-specificity phosphatase DUSP23 as a new diagnostic marker for cholangiocarcinoma, and its use for diagnosis or prognosis prediction. Furthermore, the present invention confirmed that a therapeutic effect on cholangiocarcinoma occurs when DUSP23 expression is inhibited, and confirmed that this is due to the inhibition of YAP protein expression and the inhibition of YAP phosphorylation. As such, it can be usefully utilized for the diagnosis, prognosis prediction, or treatment of cholangiocarcinoma, and thus its industrial applicability is recognized.

Claims

1. A method for providing information for the diagnosis or prognosis prediction of cholangiocarcinoma, comprising the following steps: (S1) A step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a subject; (S2) A step of comparing the protein or mRNA expression level of the above-mentioned DUSP23 with the expression level in a biological sample isolated from a control group; and (S3) A step in which, if the level of protein or mRNA expression of DUSP23 in a biological sample isolated from a subject is increased compared to the level of protein or mRNA expression of DUSP23 in a biological sample isolated from a control, the subject is determined to have cholangiocarcinoma or have a poor prognosis.

2. In Paragraph 1, The above-mentioned cholangiocarcinoma is intrahepatic cholangiocarcinoma, and A method of providing information in which the above intrahepatic cholangiocarcinoma includes early intrahepatic cholangiocarcinoma.

3. In Paragraph 1, A method of providing information in which the biological sample is any one selected from the group consisting of bile duct tissue, blood, serum, whole blood, plasma, urine, saliva, tissue, cell, trachea, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen.

4. In Paragraph 1, A method of providing information in which the above control group is a normal individual or a liver cancer individual.

5. A composition for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising as an active ingredient a preparation for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

6. In Paragraph 5, A composition for diagnosing or predicting the prognosis of cholangiocarcinoma, wherein the above-mentioned preparation is any one selected from the group consisting of a primer set, a probe, an antibody or an antigen-binding fragment thereof, a ligand, an aptamer, a peptide, a protein, a compound, an agonist, an antagonist, and a reporter vector.

7. A kit for diagnosing or predicting the prognosis of cholangiocarcinoma, comprising the composition for diagnosing or predicting the prognosis of cholangiocarcinoma according to claim 5, and instructions.

8. A screening method for agents for the prevention or treatment of cholangiocarcinoma, comprising the following steps: a) a step of measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23) in a biological sample isolated from a cholangiocarcinoma model administered a candidate substance; and b) A step of selecting the candidate substance as a preventive or therapeutic agent for cholangiocarcinoma if the protein or mRNA expression level of DUSP23 in the isolated biological sample decreases.

9. A composition for screening agents for the prevention or treatment of cholangiocarcinoma, comprising as an active ingredient a preparation for measuring the protein or mRNA expression level of DUSP23 (Dual Specificity Phosphatase 23).

10. A kit for screening agents for the prevention or treatment of cholangiocarcinoma, comprising the composition for screening agents for the prevention or treatment of cholangiocarcinoma according to claim 9, and instructions.

11. A pharmaceutical composition for the prevention or treatment of cholangiocarcinoma, comprising as an active ingredient an inhibitor of the expression or activity of DUSP23 (Dual Specificity Phosphatase 23).

12. In Paragraph 11, A pharmaceutical composition for the prevention or treatment of cholangiocarcinoma, wherein the expression or activity inhibitor is one or more selected from the group consisting of siRNA, miRNA, shRNA, dsRNA, ASO (antisense oligonucleotide), CRISPR / CAS, compounds, peptides, aptamers, polynucleotides, antibodies, protacs, small molecules, and recombinant vectors.

13. In Paragraph 11, A pharmaceutical composition for the prevention or treatment of bile duct cancer, wherein the above composition is characterized by one or more selected from the group consisting of the following: Decrease the protein level of YAP (yes-associated protein 1); Does not affect YAP mRNA levels; Increases the phosphorylation of YAP; Inhibiting the movement of YAP to the nucleus; and Inhibits the proliferation of bile duct cancer cells.

14. A kit for the prevention or treatment of cholangiocarcinoma, comprising the pharmaceutical composition for the prevention or treatment of cholangiocarcinoma according to claim 11 and instructions.

15. A method for preventing or treating cholangiocarcinoma, comprising the step of administering a pharmaceutically effective amount of a composition containing an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient to an individual in need thereof.

16. Use of a composition containing an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient for the prevention or treatment of cholangiocarcinoma.

17. Use for manufacturing a preparation for the prevention or treatment of cholangiocarcinoma comprising a composition containing an expression or activity inhibitor of DUSP23 (Dual Specificity Phosphatase 23) as an active ingredient.

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