WO2019235729A1 - Biomarker composition for predicting solid cancer prognosis comprising phosphorylated tfcp2l1 and use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for predicting a solid cancer prognosis comprising a phosphorylated TFCP2L1 protein and a use thereof, wherein as the amount of the phosphorylated TFCP2L1 protein increases, the prognosis of a solid cancer patient is significantly worsened, and thus, it is possible to use the present invention for personalized therapy in anticancer treatment, and it is also possible to use the present invention in a screening method for developing therapeutic agents from solid cancer cells.

Description

Biomarker composition for predicting solid cancer prognosis comprising phosphorylated TFCP2L1 and use thereof

The present invention relates to a biomarker composition for predicting solid cancer prognosis comprising phosphorylated transcription factor CP2-like protein 1 (TFCP2L1) and its use.

Embryonic development and homeostatic development of adult tissues, by the number of stem cells (SCs), produce differentiated precursors that undergo self-renewal / maintenance of the stem cell's own pool or are alternative cells used throughout life. By adjusting. Transcription-factors (TFs) and chromatin regulatory proteins play an important role in regulating the core functions of stem cells from embryonic stage to adulthood by maintaining specific gene expression patterns. Indeed, to maintain unique pluripotent properties, embryonic stem cells (Embyonic SCs) express certain key transcription factors such as OCT4, NANOG, and SOX2, which stop expression in differentiated somatic cells. These transcription factors enhance gene expression to keep pluripotent stem cells in an undifferentiated state and, conversely, inhibit differentiation-induced transcription to form a pluripotent core circuit. As expected, expression of pluripotency associated transcription factors is greatly inhibited once tissue development is complete.

In particular, many genes, such as STAT3, E-RAS, cMYC, KLF4, and β-catenin, which maintain embryonic stem cell phenotype in tumors or contribute to the rapid proliferation of embryonic stem cells in culture, are frequently abnormally activated for a long time Has been discovered. Therefore, studying specific mechanisms of strict regulation of these transcription factors and signaling in embryonic stem cells and adult stem cells may enhance understanding of the exact molecular nature of stem cells and pathological diversity such as aging and carcinogenesis. will be.

Meanwhile, Tfcp2l1, a member of the transcription factor CP2 family, has been identified as a WNT reactive gene in mouse embryos. Expression of Tfcp2l1 was transiently found in the inner cell population of the blastocyst and was immediately downregulated after transplantation. In this early embryonic development phase, Tfcp21111 plays a key role in maintaining the pluripotent naive. Based on the analysis of changes in the transcriptome profile, it was found that expression of TFCP2L1 is characteristically upregulated when human embryonic stem cells are converted to complete state by overexpression of KLF2, KLF4, OCT4, and murine and human embryos. The loss of pluripotency-associated transcription factors has been reported in stem cells. Mechanically, several pluripotency associated genes are targeted by TFCP2L1 , and transcription of TFCP2L1 is regulated by histone dimethylase pluripotency factors required for pluripotency, such as Oct4, Nanog and Jmjd1a. In addition, TFCP2L1 has been identified as a member of the Oct4-centric protein interaction network.

Unlike typical pluripotent transcription factors such as OCT4 and NANOG, Tfcp2l1 is expressed in the epithelium of developing or adult tissues, in particular in the exocrine glands and kidneys, which play an important role in epithelial morphogenesis, functional maturation and / or homeostasis. Indeed, 70% of Tfcp2l1- deficient mice died due to kidney hypoplasia before 5 weeks of birth. Given the association between pluripotency and WNT signaling, expression of TFCP2L1 in adult tissues is tightly regulated and limited to resident stem cell numbers. Tissue damage can lead to activation of the TFCP2L1-centric developmental pathways to regenerate damaged tissues, and excess stimulation on tissues leads to pathological conditions such as malignant tumors, which are caused by several major embryonic development genes (eg For example, STAT3, E-RAS, CMYC, KLF4, β-catenin) and signal transduction pathways (NOTCH, WNT, HEDGEHOG) have also been observed. Nevertheless, no specific molecular mechanisms regulating the expression level, activity and post-translational modification status of TFCP2L1 in adult pathological physiological conditions have yet been studied.

Bladder cancer is the most common carcinoma in US men, and among the bladder cancers, urothelial carcinoma is the most common histologic subtype in which more than 90% of patients are seen. Approximately 70-80% of patients with bladder cancer show noninvasive or early invasive (non-muscularis propria-invasive, NMIBC) disease at initial diagnosis. NMICB is not life-threatening, but consequently 50-70% of NMICB cases recur and 15-25% develop muscularis propria-invasive (MIBC), leading to distant metastases and death. It is associated with high risks such as Clinical pathologic features such as tumor stage and severity in bladder cancer are useful for risk assessment. However, only a few tools can predict clinical outcomes among patients with similar clinicopathological features. Therefore, one of the biggest challenges in controlling bladder cancer is to identify markers for predicting new prognosis of aggressive disease in customized chemotherapy.

An object of the present invention is to provide a biomarker composition for predicting solid cancer prognosis comprising phosphorylated TFCP2L1 protein or a fragment of the protein.

Another object of the present invention is to provide a composition for predicting solid cancer prognosis comprising an agent capable of detecting phosphorylated TFCP2L1 protein or a fragment of the protein.

Still another object of the present invention is to provide a kit for predicting solid cancer prognosis comprising the composition.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating solid cancer, or a health functional food composition for preventing or improving solid cancer, comprising a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or the protein fragment as an active ingredient.

It is another object of the present invention to provide a method for providing information necessary for predicting solid cancer prognosis.

It is another object of the present invention to provide a method for screening a solid cancer therapeutic agent.

Another object of the present invention is to provide the use of phosphorylated TFCP2L1 protein or fragment of said protein as a biomarker for predicting solid cancer prognosis.

Another object of the present invention is to provide a use of an agent capable of detecting phosphorylated TFCP2L1 protein or fragment of said protein for predicting solid cancer prognosis.

It is another object of the present invention to provide a use of a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or protein fragment for the manufacture of a medicament for the prevention or treatment of solid cancer.

It is another object of the present invention to provide a use of a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or protein fragment for the manufacture of a dietary supplement for preventing or improving solid cancer.

In order to achieve the above object, the present invention provides a biomarker composition for predicting solid cancer prognosis comprising a phosphorylated TFCP2L1 protein or a fragment of the protein.

The present invention also provides a composition for predicting solid cancer prognosis comprising an agent capable of detecting phosphorylated TFCP2L1 protein or a fragment of the protein.

The present invention also provides a kit for predicting solid cancer prognosis comprising the composition.

In order to achieve the above another object, the present invention provides a pharmaceutical composition for preventing or treating solid cancer, or a health functional food composition for preventing or improving solid cancer, comprising a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or the protein fragment as an active ingredient. to provide.

In order to achieve the above another object, the present invention comprises the steps of (a) measuring the amount of phosphorylated TFCP2L1 protein or protein fragments in a sample isolated from a solid cancer patient; (b) comparing the amount of the phosphorylated TFCP2L1 protein or protein fragment with a control sample; And (c) when the amount of the phosphorylated TFCP2L1 protein or the protein fragment is higher than the control sample, determining that the prognosis is poor, it provides a method for providing information necessary for predicting solid cancer prognosis.

In order to achieve the above another object, the present invention comprises the steps of (a) treating a test substance to a solid cancer cell; (b) measuring the amount of phosphorylated TFCP2L1 protein or protein fragment in the solid cancer cell; (c) comparing the amount of the phosphorylated TFCP2L1 protein or protein fragment with a control sample not treated with the test substance; And (d) when the amount of the phosphorylated TFCP2L1 protein or the protein fragment is reduced than that of the control sample, determining the test substance as a cancer treatment agent.

In order to achieve the above another object, the present invention provides the use of a phosphorylated TFCP2L1 protein or a fragment of the protein as a biomarker for the prediction of solid cancer prognosis.

In order to achieve the above another object, the present invention provides the use of a phosphorylated TFCP2L1 protein or a fragment of the protein for the prediction of solid cancer prognosis.

In order to achieve the above another object, the present invention provides the use of a phosphorylation inhibitor to inhibit the phosphorylation of TFCP2L1 protein or the protein fragment for the manufacture of a medicament for the prevention or treatment of solid cancer.

In order to achieve the above another object, the present invention provides the use of a phosphorylation inhibitor to inhibit the phosphorylation of TFCP2L1 protein or protein fragments for the manufacture of health food for the prevention or improvement of solid cancer.

The present invention relates to a biomarker for predicting solid cancer prognosis including phosphorylated TFCP2L1 and its use, wherein TFCP2L1 is phosphorylated by CDK1, and as the amount of TFCP2L1 phosphorylated increases, the prognosis of solid cancer patients is poor. In addition to predicting the prognosis of solid cancer, it can be used not only to treat cancer more effectively, but also as a screening method for new cancer treatments.

1 is a Kaplan-Meier plot of the overall survival of patients with (A) high or low expression levels of TFCP2L1 in bladder cancer cohorts obtained from the TCGA dataset and (B) normal bladder epithelial cells (HB1EpC) and two bladder cancer cell lines J82 And Western blot results of specific proteins at T24.

Figure 2 shows the results of cell proliferation (A) and tumor cell formation assays (B) in HB1EpC and T24 cell lines, respectively (n = 5, open reading frame (ORF) or shRNA for human TFCP2L1 and CDK1 were lentiviral infections) Representative illustrations of tumor sphere formation in T24 bladder cancer cells are shown at magnifications of X40 (upper panel) or X200 (lower panel) scale bar = 100 mm).

FIG. 3 shows (A) Flag- for detection of phosphorylated threonine 177 (p-Thr) in Flag-tagged TFCP2L1 (Flag-TFCP2L1) expressing cells after 5 h treatment with or without 25 uM Roscovitine. Results of immunoprecipitation (IP) analysis, (B) Flag-IP analysis for p-Thr detection in Flag-TFCP2L1 expressing cells that silenced CDK1, (C) between Flag-TFCP2L1 and CDK1 protein Flag-IP analysis results to identify the physical interaction of the.

FIG. 4 shows the results of tissue microarray analysis of TFCP2L1 (p-TFCP2L1) and CDK1 expression phosphorylated with threonine 177 measured by immunohistochemical staining in tissue microarrays including urinary resection of bladder cancer samples of 400 patients. .

FIG. 5 shows a Kaplan-Meier plot of cancer-specific viability associated with expression levels of p-TFCP2L1 (p = 0.001) and CDK1 (p = 0.002) proteins.

FIG. 6 shows a scatter plot of the H-scores of p-TFCP2L1 or CDK1 proteins in 400 patient cases (linear regression analysis shows a positive correlation (p <0.001) between p-TFCP2L1 and CDK1 protein expression). ).

FIG. 7 shows the results of opal multiplex immunofluorescence staining in tissue microarrays including transurethral resection of bladder cancer samples from 400 patients, p-TFCP2L1 (yellow), CDK1 (red) and cytokeratin (green) in bladder cancer. A representative figure is shown (nuclei are counterstained with DAPI [blue]).

FIG. 8 shows the clinicopathological correlations of p-TFCP2L1 and CDK1 protein expression and co-expression (statistical significance parameters [p <0.05]) in red. Not assessable: caused by cautery artifacts, fragmentation, or incorrect orientation of tumor tissue; PUNLMP; Papillary urinary tract epithelial neoplasm with low possibility of malignancy).

9 shows a Kaplan-Meier plot of cancer-specific survival (p <0.001) related to the co-expression ratios of p-TFCP2L1 and CDK1 proteins in the bladder cancer cohort of the present invention.

10 shows the results of univariate and multivariate analysis of cancer-specific survival rates.

FIG. 11 is a tissue microarray analysis (TMA) of bladder cancer stem cell markers SALL4 and CD44 expression in p-TFCP2L1 ⁺ cells of 400 bladder cancer patients, more specifically cytokeratin during opal multiple fluorescent immunostaining in bladder cancer. Green), p-TFCP2L1 (yellow), CDK1 (red), CD44 (green) and SALL4 (orange) are shown (the nucleus is counterstained with DAPI [blue] and the result).

FIG. 12 is a scatter plot of the median value of the quadrant (n = 322, *** p <0.001) of (A) the proportion of CDK1, SALL4 and CD44 expressing cells among cells expressing p-TFCP2L1 high or low. Shown and (B) is a schematic overview of the proposed model.

FIG. 13 shows high TFCP2L1 expression (red) level or low TFCP2L1 expression level (black) in patients with gastric cancer, breast cancer, uterine cancer, lung cancer, colon cancer, kidney cancer, liver cancer, melanoma and glioma using KM plotter database Survival rate based on

FIG. 14 shows representative results of tumor cell formation assays in MCF7 cells, breast cancer cell lines (A; X40, X100, or X200 magnification; scale bar = 100 mm), and quantitative data thereof (B; all data Mean ± SEM [n = 5]; compared to the empty control group, *** p <0.001, two-variate analysis was performed with Bonferroni post-mortem analysis and ORF of human TFCP2L1 and CDK1 was determined by lentiviral. Infected and delivered), and the expression levels of total TFCP2L1 (t-TFCP2L1) or phosphorylated TFCP2L1 (p-TFCP2L1) and CDK1 protein were analyzed by Western blot analysis (C).

Figure 15 (A) Immunohistochemical results for p-TFCP2L1, SOX2 and SALL4 stem cell marker proteins in tissue pellet blocks prepared from human teratocarcinoma cells (NTERA2) and differentiated primary fibroblasts (IMR90) (tissues) All antibodies used in the immunoassay of microassays are specific for antigens obtained from NTERA2, and commercially available TFCP2L1 antibodies (Aviva Systems Biology; OAAB09732) were excluded because they showed little signal upon NTERA2 staining), and (B) immunohistochemical staining results of p-TFCP2L1 and CDK1 in normal urinary tract epithelial samples.

FIG. 16 shows (A) Receiver operating characteristic curve (ROS) and (ROS) for p-TFCP2L1 or CDK1 expression in a tissue microassay structure obtained by performing a urethral resection of a tumor sample of 400 bladder cancer patients. B) Receptor action characteristic curves for co-expression of p-TFCP2L1 and CDK1 proteins.

Figure 17 shows a representative representation of opal multiple immunofluorescence staining of cancer stem cell markers p-TFCP2L1, CDK1, SALL4 and CD44.

18 shows sequence alignment of human TFCP2L1.

Figure 19 shows the Gene Ontology (GO) category and corresponding genes used in the experiment.

FIG. 20 shows the results of Western blot analysis using polyclonal antibodies specific for Thr177 (p-Tfcp2l1 (T177)) of phosphorylated Tfcp2l1 for mESC transfected with Flag-tagged or untagged Tfcp2l1 expression plasmid (The specificity of P-Tfcp2l1 [T177] was confirmed by competition assay with 2 mg phospho-peptide antigen during the blocking process, and Tfcp2l1-T177A mutant expressing cells showed little signal [left panel]. And the amino acid sequence of the peptide antigen representing human Tfcp2l1 is shown below and the T177 modified by phosphorylation in the peptide antigen is shown in red The expression of the Tfcp2l1 protein was confirmed using Flag and Tfcp2l1 specific antibodies [right panel] Β-actin was used as an intrinsic control; WB; Western blot).

The inventors of the present invention found that in patients with bladder cancer, accidental coactivation of the CDK1 and TFCP2L1 cascades is associated with high tumor grade, lymphovascular invasion, and endocrine invasion, frequent metastasis to distant organs, and short patient survival. It has been confirmed that the stimulation of cancer stem cell activity associated with aggressive clinicopathological parameters including the present invention and completed the present invention.

Accordingly, the present invention provides a biomarker composition for predicting solid cancer prognosis comprising phosphorylated TFCP2L1 protein or a fragment of the protein.

In one embodiment of the present invention, the TFCP2L1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, the phosphorylated TFCP2L1 protein may be a phosphorylated 177 th threonine of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto Does not.

In addition, the fragment of the phosphorylated TFCP2L1 protein may be phosphorylated 17th threonine of the amino acid sequence of SEQ ID NO: 2 or 7 threonine of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

The TFCP2L1 protein or protein fragment may be phosphorylated by CDK1, but is not limited thereto.

In this case, when the amount of the phosphorylated TFCP2L1 protein or the protein fragment is increased, it can be predicted that the prognosis of the solid cancer is not good.

The solid cancer may be one or more selected from the group consisting of bladder cancer, colorectal cancer, gastric cancer, and kidney cancer, but is not limited thereto.

As used herein, the term “prognosis” refers to the prospect of future symptoms or progression determined by diagnosing a disease. Prognosis prediction is a very important clinical task as it provides clues to the direction of future chemotherapy.

In addition, the present invention provides a composition for predicting solid cancer prognosis comprising an agent capable of detecting phosphorylated TFCP2L1 protein or a fragment of the protein.

The TFCP2L1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, the phosphorylated TFCP2L1 protein may be a phosphorylated 177 th threonine of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In addition, the fragment of the phosphorylated TFCP2L1 protein may be phosphorylated 17th threonine of the amino acid sequence of SEQ ID NO: 2 or 7 threonine of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

In this case, the agent may be an antibody, peptide, aptamer or compound that specifically binds to the protein, but is not limited thereto.

The present invention also provides a kit for predicting solid cancer prognosis comprising the composition.

As used herein, the term “antibody” refers to a specific immunoglobulin directed to an antigenic site as is known in the art. An antibody in the present invention means an antibody that specifically binds to the phosphorylated TFCP2L1 or phosphorylated TFCP2L1 fragment of the present invention, and the antibody may be prepared according to conventional methods in the art. Forms of such antibodies include polyclonal antibodies or monoclonal antibodies, including all immunoglobulin antibodies. The antibody means a complete form having two full length light chains and two full length heavy chains. In addition, the said antibody also contains special antibodies, such as a humanized antibody.

As used herein, the term "peptide" has the advantage of high binding power to the target material, and no degeneration occurs even during thermal / chemical treatment. In addition, the small size of the molecule can be used as a fusion protein by attaching to other proteins. Specifically, since it can be used by attaching to a polymer protein chain, it can be used as a diagnostic kit and drug delivery material.

As used herein, the term "aptamer" refers to a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and has the ability to bind to a target molecule with high affinity and specificity. It means a kind of polynucleotide consisting of). As described above, aptamers are composed of polynucleotides that can bind specifically to antigenic substances like antibodies, but are more stable than proteins, simple in structure, and easy to synthesize. Can

In addition, the kit of the present invention includes an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that is developed by reaction with a substrate, a color substrate solution to be color-reacted with the label, a wash solution, It may include an enzyme stopping solution and the like, and may be prepared in a number of separate packaging or compartments containing the reagent components used.

In addition, the present invention provides a pharmaceutical composition for preventing or treating solid cancer, comprising a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or the protein fragment as an active ingredient.

The TFCP2L1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, and the phosphorylation may be performed at 177 threonine of the TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The phosphorylation may be at 17 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or at 7 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

In one embodiment of the invention, the phosphorylation inhibitor may be an antibody, but is not limited thereto.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the phosphorylation inhibitor, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants. Solubilizers, such as an agent or a flavoring agent, can be used.

The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Pharmaceutical formulation forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be. The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered.

Suitable dosages of the pharmaceutical compositions of the present invention may be appropriately selected by those skilled in the art, depending on the condition and weight of the patient, the extent of the disease, the form of the drug, and the time, and may be administered once to several times daily if necessary. have.

In another aspect, the present invention provides a health functional food composition for preventing or improving solid cancer comprising a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or the protein fragment as an active ingredient.

The TFCP2L1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, and the phosphorylation may be performed at 177 threonine of the TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The phosphorylation may be at 17 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or at 7 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

In one embodiment of the invention, the phosphorylation inhibitor may be an antibody, but is not limited thereto.

The dietary supplement composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. Others may contain pulp for the production of natural fruit juices, synthetic fruit juices and vegetable drinks. These components can be used independently or in combination. In addition, the nutraceutical composition is in the form of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverages, tea, functional water, drinks, alcohol and vitamin complexes Can be.

In addition, the health functional food composition may further include a food additive, the suitability as a "food additive" is the item according to the General Regulations and General Test Methods of the Food Additives Code approved by the Food and Drug Administration unless otherwise specified It is determined by the standard and the standard.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, cinnamon acid, natural additives such as color pigments, licorice extract, crystalline cellulose, high-quench pigments, guar gum, L Mixed preparations, such as a sodium glutamate preparation, a noodles addition alkali agent, a preservative preparation, and a tar pigment preparation, etc. are mentioned.

Moreover, the present invention provides a method for preparing a human cancer cell, comprising: (a) measuring the amount of phosphorylated TFCP2L1 protein or protein fragment in a sample isolated from a solid cancer patient; (b) comparing the amount of the phosphorylated TFCP2L1 protein or protein fragment with a control sample; And (c) when the amount of the phosphorylated TFCP2L1 protein or the protein fragment is higher than the control sample, determining that the prognosis is poor, it provides a method for providing information necessary for predicting solid cancer prognosis.

In addition, the present invention comprises the steps of (a) treating a test substance to a solid cancer cell; (b) measuring the amount of phosphorylated TFCP2L1 protein or protein fragment in the solid cancer cell; (c) comparing the amount of the phosphorylated TFCP2L1 protein or protein fragment with a control sample not treated with the test substance; And (d) when the amount of the phosphorylated TFCP2L1 protein or the protein fragment is reduced than that of the control sample, determining the test substance as a cancer treatment agent.

The TFCP2L1 protein may be composed of the amino acid sequence of SEQ ID NO: 1, and the phosphorylation may be performed at 177 threonine of the TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The phosphorylation may be at 17 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or at 7 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

Specifically, as a method for measuring the amount of the phosphorylated TFCP2L1 protein or the protein fragment, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion) ), Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chips, It is not limited to this.

As used to refer to the screening methods of the present invention, the term "test material" refers to an unknown candidate used in screening to test whether it affects the phosphorylation of TFCP2L1 protein or the protein fragment. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

The solid cancer may be one or more selected from the group consisting of bladder cancer, colorectal cancer, gastric cancer, and kidney cancer, but is not limited thereto.

The present invention also provides the use of phosphorylated TFCP2L1 protein or fragment of said protein as a biomarker for predicting solid cancer prognosis.

The present invention also provides the use of an agent capable of detecting phosphorylated TFCP2L1 protein or fragment of said protein for predicting solid cancer prognosis.

The present invention also provides the use of a phosphorylation inhibitor that inhibits phosphorylation of a TFCP2L1 protein or protein fragment for the manufacture of a medicament for preventing or treating solid cancer.

The present invention also provides the use of a phosphorylation inhibitor that inhibits the phosphorylation of TFCP2L1 protein or protein fragments for the manufacture of a dietary supplement for the prevention or improvement of solid cancer.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are provided to more fully explain the present invention to those skilled in the art, and the scope of the present invention is not limited to the following examples, only to illustrate the contents of the present invention. no.

Example 1 Confirmation of Use of TFCP2L1 as a Marker for Predicting Bladder Cancer Prognosis

1. Experiment Method

1) Cell Culture

Human HBlEpC Primary Bladder Epithelial Cells (Cell Applications, Inc, San Diego, Calif.), J82 and T24 Bladder Cancer Cell Lines (ATCC), Breast Cancer Cell Line MCF7 (ATCC), and Human Teratoma Cell Lines NTERA2 (ATCC), IMR900 Primary Fibroblasts ( ATCC) were cultured under the following conditions: HBlEpC derived from normal human bladder (Cell Applications, Inc, San Diego, Calif.) Was cultured in bladder epithelial cell growth medium (Cell Applications, Inc). The culture medium used in the culture was Dulbecco's modified Eagle's (Dulbecco's modified Eagle's) supplemented with penicillin / streptomycin (Cellgro) and 10% heat inactivated fetal bovine serum (FBS; Hyclone). . In addition, the human bladder cancer cell lines J82 and T24 (ATCC, Manassas, Pittsburgh, PA) were incubated in McCoy's 5A medium (MCCoy's 5A medium; ATCC) with CO ₂ incubators at 37 ° C. and 5% daily, replacing the medium. It was.

2) Proteomic analysis for identification of TFCP2L1 interactor and phosphorylation sites

The total amino acid sequence of human TFCP2L1 protein is shown in FIG. 18.

TFCP2L1 interacting protein complexes were prepared by immunoprecipitation (IP) experiments. Specifically as follows: For IP analysis, the cell extract was subjected to IP lysis buffer (50 mM Tris-Cl [pH 7.4], 0.5% NP-40, 150 mM NaCl, 1.5 mM MgCl ₂ , supplemented with protease / protease activity). After washing with 2 mM DTT, 2 mM EGTA) and phosphatase inhibitor mixture (Roche), it was centrifuged (12,000 g, 10 min at 4 ° C.). Then ANTI-FLAG® M2 magnetic beads (Sigma-Aldrich) or protein mixed with 1 μg antibody for 3 hours at 4 ° C. in the presence or absence of 200 units of Benzonase Nuclease in the cell extract. G magnetic beads (Millipore, Sigma-Aldrich) were treated. This immunoprecipitated protein was washed four times with IP lysis buffer, and the bound protein was eluted with 3x FLAG peptide (Sigma-Aldrich) or 0.1M glycine-hydrochloric acid (HCl) buffer (pH 3.0) according to the manufacturer's instructions.

The complex was reduced to dithiothreitol and alkylated iodoacetamide and degraded with trypsin at 37 ° C. for 16 hours. Peptide mixtures were desalted into solid phase extracts using C18 cartridges and analyzed on a mass spectrometer (Thermo Scientific LTQ Orbitrap-XL) connected to a nano LC system (Shimadzu, Japan). Raw files were processed into mgf files using user written script for identification, ProteinPilot Software (AB Sciex, Framingham, MA, USA version 5.0) and CompPASS algorithm. Sowa et al., 2009) were used to find data in the UniProt target-decoy sequence database containing common contaminant proteins. 2% false discovery rate (FDR) in protein identification was determined using a Posterior Error Probability (PEP) algorithm. The dataset of the proteomics analysis was as shown in Table 1 below.

3) Definition of TFCP2L1 Interaction Complexes and Transcription Targets

Whole-gene transcription targets of TFCP2L1 were previously reported in the TFCP2L1 ChIP-seq dataset (GED11431; Chen, X. et al., Integration of External Signaling Pathways with the Core Transcriptional) with a cutoff value (≥0.5 related score). Network in Embryonic Stem Cells. Cell 133 , 1106-1117). Functional analysis of the TFCP2L1 proteomics and ChIP-seq datasets related to genetic networks, biofunctions and canonical pathways was performed using Metacore (Clarivate Analytics, Philadelphia, PA) microarray software with default values set. . Specific Gene Ontology (GO) categories and corresponding genes were as shown in FIG. 19.

4) Human Bladder Cancer Research Sample

This retrospective study was approved by the Institutional Review Board of Asan Medical Center (AMC; 2013-107). The human sample of this study was used for tissue microarray (TMA) structure after undergoing transurethral resection of bladder tumor (TURBT) at Asan Medical Center from January 1996 to December 2006. 403 patients with possible cancerous tissue were included. Clinical information, including patient tumor recurrence, distant tissue metastasis and survival rates, was obtained from an electronic medical record on a hospital chart, and initial and recurrent tumors were assessed to assess histological tumor stage according to diagnostic reassessment and the 2016 World Health Organization classification. All pathological data included were reviewed. TNM classification steps were assigned according to the eighth edition of the American Joint Committee on Cancer Staging System (AJCC).

In addition, a more specific experimental method for tissue microarray (TMA) was as follows: Bladder cancer patient TMA block was analyzed using a tissue microarray instrument (Beecher Instruments, Silver Spring, MD), where the bladder cancer patient TMA block was Resection (TURBT) specimens were made from urinary epithelial bladder cancer tissue blocks embedded in paraffin after being fixed with 10% neutral buffered formalin. To overcome the problem of tumor dysplasia, blocks were prepared to include three representative cores in different regions of each tumor. Blocks were also prepared from tissues of cervical cancer (control 1) and colon cancer (control 13) animals and normal urinary valves (control 14) with bladder removed during hysterectomy or colon resection.

Immunohustochemistry (IHC) staining and Opal multiplexed IF staining methods were as follows: TMA slides were evaluated by two independent pathologists (Byung-Boo Jo and Cho Mi-mi), with TMA slides After IHC and opal multiple immunofluorescence staining, the nuclear positive rate of p-TFCP2L1 and the nuclear and cytoplasmic positive rate of CDK1 in cancer cells were recorded, respectively. Staining intensities were categorized into four stage systems: negative (0), weak (1+), normal (2+) or strong (3+).

Pathological and immunohistochemical evaluation and H-scoring as shown in FIGS. 16A and 16B for protein expression were performed as follows: Staining intensity of cancer cells (scores 0-3 +) multiplied by percentage of immune-responsive cells. H scores between 0 and 300 were obtained and indicated.

In addition, tumor cell formation assays were performed as follows: First, a single cell suspension of bladder cancer cells isolated from bladder cancer patients was prepared, and serum-free keratinocyte growth media (Geratinocyte Growth Media; Gibco, Waltham, Mass.) After resuspending the growth factor-reducing Matrigel (BD Biosciences, Mountain View, Calif.) In a 1: 1 ratio, the cell suspension and the resuspended medium solution were ultra low attachment plate (Costar, Corning, NY). Single cancer cells were cultured. Tumor sphere formation was then analyzed 7 days after the first incubation, with image J software (National Health, Bethesda) demarcating the size of the embryoid body or tumor sphere from eight representative regions randomly selected from each group. , MD).

Finally co-expression subgrouping was performed as follows: the proportion of cells co-expressing p-TFCP2L1 and CDK1 was recorded and in each case using receiver operating characteristic (ROC) curve analysis. Based on the cut-off points calculated, the results were classified into high co-expression versus low co-expression.

5) Statistical analysis

In vitro molecular and cell biology data were analyzed by one-way ANOVA or two-way ANOVA with Bonferroni's post-hoc analysis. In addition, GraphPad Prism 6.0 (GraphPad Software, La Jolla, Calif.) Was used to perform all analyzes.

In the bladder cancer patient study, statistical analysis was performed using SPSS version 12.0, and Pearson's Chi-square and Fisher's exact tests to determine the correlation between tested antibodies and clinicopathological parameters. (Fisher's exact) test was performed. In addition, univariate survival analysis was performed to confirm the clinical significance of antibody expression and clinicopathologic parameters, and through multivariate analysis, the Cox proportional hazards model used 95% confidence interval for each factor ( confidence intercal (CI) was used to assess hazard ratio (HR). All experiments were performed with two-sided tests, and P values of 0.05 or less were considered statistically significant.

2. Experimental Results

1) TFCP2L1 as a Biomarker with an Unfavorable Prognosis in Urinary Epithelial Cell Carcinoma

First, we tried to confirm the clinical significance of TFCP2L1 phosphorylation on tumor formation in the epithelial tissue of bladder where abnormal expression of TFCP2L1 occurs in adulthood. Thus, using a published database of Kaplan-Meier plotters (http://kmplot.com/) or Cancer Genome Atlas Cancer Genome (TCGA), bladder cancer and in particular, as shown in Figures 1A and 13, Patients with high TFCP2L1 expression among gastric cancer, colorectal cancer and kidney cancer patients were found to have a short overall survival rate. In contrast, there was no significant relationship between TFCP2L1 expression and survival in patients with liver cancer, melanoma and glioma. Furthermore, when compared to primary human bladder epithelial cells (HBlEpC), the expression levels of TFCP2L1 and CDK1 proteins and transcription targets such as KLF2 and KLF4 in both bladder cancer cell lines such as J82 and T24, as shown in FIG. Although very high, OCT4 and NANOG proteins were hardly expressed.

In addition, as shown in FIG. 2A, cell proliferation of HB1EpC was stimulated by ectopic expression of TFCP2L1 and further enhanced by coexpression of TFCP2L1 and CDK1, but this proliferation was inhibited when they were silenced. In addition, as shown in FIG. 2B, the tumor cell formation ability of bladder cancer cells was severely impaired by TFCP2L1 and CDK1 silencing, and as shown in FIGS. 14A-14C, even in tumor cell formation assays using the breast cancer cell line MCF7. Similar results were observed.

2) Confirmation of phosphorylation of threonine 177 of TFCP2L1 by CKD1

Next, the phosphorylation of TFCP2L1 by CDK1 in bladder cancer was confirmed (p-TFCP2L1) based on the fact that the threonine position 177 (Thr177) of TFCP2L1 proteins was very well conserved in several species. That is, immunoprecipitation analysis of TFCP2L1 tagged with Flag in T24 cells was performed. As a result, as shown in FIGS. 3A and 3B, it was confirmed that a significant amount of TFCP2L1 proteins were phosphorylated depending on the expression or activity of CDK1. there was. In addition, as shown in FIG. 3C, TFCP2L1 and CDK1 proteins physically interacted in T24 cells, demonstrating the functional pathway of CDK1-TFCP2L1 in bladder cancer.

3) Check the relationship between bladder cancer progression and TFCP2L1 and CDK1 proteins

In bladder cancer progression, tissue microarray structures formed from TURBT samples from 400 patients were identified and correlated with clinical results to confirm the expression level and co-expression function of phosphorylated TFCP2L1 and CDK1 proteins. Was evaluated. In addition, for the polyclonal antibody specific for p-TFCP2L1 (T177) protein, an antibody targeting the amino acid sequence of Table 2 was prepared, as shown in Figure 20, Western blot was performed.

Amino acid sequence SEQ ID NO: KRASAFIQVH CISTEFTPRK HGGEKGVPFR VQIDTFKQNE 2 CISTEFTPRK HG 3

At this time, the clinical and pathological characteristics of all 400 cases are listed in Tables 3 and 4 below.

As a result, as shown in FIG. 15A and Table 5 below, in immunofluorescence (IF) staining of cell blocks obtained from the human teratocarcinoma cell line NTERA2, the p-TFCP2L1 antibody was specifically specific for the p-TFCP2L1 protein. It was detected as a target, but not in IMR900 primary fibroblasts.

In addition, referring to FIG. 15B, p-TFCP2L1 and CDK1 proteins were expressed only in small cells of the basal and subbasal layers of normal urinary epithelial cells, indicating an average of 10.0 H-scores. In contrast, in the majority of bladder cancer samples, as shown in FIG. 6, a very wide range of H-scores appeared (p-TFCP2L1; median of 87.7, 0.0-231.0. CDK1; median of 100.0, 0.0- 291.0), as shown in FIG. 4, the expression of p-TFCP2L1 and CDK1 was significantly increased (92.8%, 371/400 and 93.0%, 372/400, respectively), as shown in FIGS. 1B and 2. In vitro obtained from the cell lines were consistent with the experimental results.

Referring next to FIG. 5, p-TFCP2L1 or CDK1 proteins were expressed very high in tumor samples with aggressive clinicopathological characteristics, and the patient group exhibiting this high expression showed a very short cancer specific survival rate.

Accordingly, high expression of p-TFCP2L1 was shown in Figure 8, as shown in Figure 8, high tumor degree (p = 0.002), high tumor stage (p = 0.022), pT category (p = 0.022), LVI (p-0.042) , Intrinsic muscle layer invasion (p-0.026) and cancer-specific death (p-0.01). In addition, the expression level of high CDK1 was also high in tumor stage (p <0.001), pT category (p = 0.018), LVI (p = 0.023), eigenmuscular invasion (p = 0.011), distant metastasis (p = 0.016) and cancer. It was associated with specific survival (p = 0.001).

4) Identify the clinical significance of TFCP2L1 and CDK1 coexpression in bladder cancer

As described above, the expression of p-TFCP2L1 and CDK1 proteins showed a positive correlation with each other (p <0.001, see FIG. 6). Thus, to confirm the clinical significance in bladder cancer of such co-expression, as shown in FIG. 7, opal multiple immunofluorescence staining was performed on bladder cancer tissues collected from bladder cancer patients labeled with cytokeratin (CK). As a result, as shown in FIG. 8, high co-expression of p-TFCP2L1 and CDK1 resulted in high tumor stage (p <0.001), eigenmuscular invasion (p-0.041), distant metastasis (p = 0.040) and cancer specific. Significantly associated with aggressive clinical pathologic features associated with death (p <0.001). In addition, as shown in FIG. 9, this coexpression was associated with a low cancer-specific survival rate (p <0.001), and as shown in FIG. 10, in multivariate analysis, in addition to age, lymph node metastasis and intrinsic muscle layer invasion, It was also associated with an independent prognostic factor (p = 0.04) for cancer-specific survival.

In addition, as shown in FIGS. 11 and 17, opal multiple immunofluorescence staining revealed that the p-TFCP2L1 ⁺ bladder cancer cells in the tissues of bladder cancer patients had many reported markers of cancer stem cells (Cancer Stem cells) including SALL4 and CD44. It was confirmed that the expression. As shown in FIG. 12, SALL4 and CD44 proteins were simultaneously located in cancer cells showing high p-TFCP2L1 levels, as shown in FIG. 12. From these results, p-TFCP2L1 ⁺ It was found that the cells could represent cancer stem cell populations.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (21)

A biomarker composition for predicting solid cancer prognosis comprising a phosphorylated TFCP2L1 protein or a fragment of the protein. The biomarker composition of claim 1, wherein the phosphorylated TFCP2L1 protein is phosphorylated with 177 threonine in the amino acid sequence of SEQ ID NO: 1. The biomarker composition according to claim 1, wherein the fragment of the protein is phosphorylated with 17 th threonine in the amino acid sequence of SEQ ID NO: 2 or with th th thionine in the amino acid sequence of SEQ ID NO: 3. The biomarker composition of claim 1, wherein the solid cancer is at least one selected from the group consisting of bladder cancer, colorectal cancer, gastric cancer, and kidney cancer. A composition for predicting solid cancer prognosis comprising an agent capable of detecting phosphorylated TFCP2L1 protein or a fragment of the protein. The composition of claim 5, wherein the phosphorylated TFCP2L1 protein is phosphorylated with 177 threonine in the amino acid sequence of SEQ ID NO: 1. The composition of claim 5, wherein the fragment of the protein is phosphorylated with 17 th threonine in the amino acid sequence of SEQ ID NO: 2 or with th th thionine in the amino acid sequence of SEQ ID NO: 3. 7. 6. The composition for predicting solid cancer prognosis according to claim 5, wherein the agent is an antibody, peptide, aptamer or compound that specifically binds to the protein or protein fragment. A kit for predicting solid cancer prognosis comprising the composition according to claim 5. A pharmaceutical composition for preventing or treating solid cancer, comprising a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or the protein fragment as an active ingredient. The pharmaceutical composition for preventing or treating solid cancer according to claim 10, wherein the phosphorylation is at 177 threonine of the TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 10, wherein the phosphorylation is in the 17 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or in the 7 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 3 Or therapeutic pharmaceutical compositions. A health functional food composition for preventing or improving solid cancer, comprising a phosphorylation inhibitor that inhibits phosphorylation of TFCP2L1 protein or the protein fragment as an active ingredient. The health functional food composition for preventing or improving solid cancer according to claim 13, wherein the phosphorylation is at 177 threonine of the TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 13, wherein the phosphorylation is at the seventh threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or the 17 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 Or improved dietary supplement composition. (a) measuring the amount of phosphorylated TFCP2L1 protein or protein fragment in a sample isolated from a solid cancer patient; (b) comparing the amount of the phosphorylated TFCP2L1 protein or protein fragment with a control sample; And (c) determining that the prognosis is poor when the amount of phosphorylated TFCP2L1 protein or the protein fragment is higher than that of the control sample. Including, the method for providing information necessary for the prediction of solid cancer prognosis. 17. The method of claim 16, wherein said phosphorylation is at 177 threonine of the TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1. 17. The prognosis of solid cancer according to claim 16, wherein the phosphorylation is at the seventh threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or the seventeen threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 How to provide the information needed for forecasting. (a) treating the test substance with solid cancer cells; (b) measuring the amount of phosphorylated TFCP2L1 protein or protein fragment in the solid cancer cell; (c) comparing the amount of the phosphorylated TFCP2L1 protein or protein fragment with a control sample not treated with the test substance; And (d) when the amount of the phosphorylated TFCP2L1 protein or protein fragment is reduced than the control sample, judging the test substance as a cancer treatment agent Screening method for the treatment of solid cancer, comprising. 20. The method of claim 19, wherein said phosphorylation is at the 177 th threonine of TFCP2L1 protein consisting of the amino acid sequence of SEQ ID NO: 1. 20. The therapeutic agent for solid cancer according to claim 19, wherein the phosphorylation is at 17 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 2 or 7 th threonine of the TFCP2L1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 3 Screening method.

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