KR101994821B1 - Use of forkhead box o 3 protein - Google Patents

Abstract

According to the present invention, the anticancer drug screening, anticancer therapy and anticancer therapy prognosis prediction are predicted using the glycosylation of FOXO3 protein (FOXO3), particularly FOXO3 protein, to improve the effect of chemotherapy, .

Description

USE OF FORKHEAD BOX O 3 PROTEIN -

The present invention relates to the use of FOXO3 protein (FOXO3), particularly FOXO3 protein, for the prediction of prognosis after chemotherapy screening, chemotherapy and chemotherapy using the glycosylation of FOXO3 protein.

According to the American Cancer Society, there are 1.66 million new cases each year and 580,000 deaths per year from cancer-related factors. Pancreatic cancer is the 9th most common cancer in the world and is the fourth most common malignancy (American Cancer Society, Cancer facts and figures, 2016). Pancreatic cancer has a 5-year survival rate of less than 5%, making it difficult to diagnose early, and more than 80% of patients are found at the stage of tumor progression (Lahoud MJ, et al., World J Gastrointest Oncol. , 599-606, 2016).

Pancreatic cancer is the fifth most common cancer in Korea, with a 5 - year survival rate of 7 to 8% and a very low survival rate compared with other cancers. Pancreatic cancer does not have an accurate early diagnosis marker, and there are no symptoms until it progresses significantly. Once pancreatic cancer is diagnosed, surgery is often impossible. Even if surgery is possible, 80 to 90% of patients who have undergone surgery will recur and die.

Liver cancer is the sixth most common cancer worldwide, mostly in Africa and Asia, and the fourth highest mortality rate. More than 95% of patients die of cancer within five years after diagnosis. Liver cancer is accompanied by pain jaundice, poor appetite, and weight loss.

Gastric cancer is a cancer that causes malignant ulcer due to adenocarcinoma in gastrointestinal mucosal tissues. It is still the second most common cause of cancer - related death in developed countries.

Forkhead box O 3 (FOXO3), on the other hand, is the transcription factor that acts at the lowest level of the insulin-like growth factor signaling pathway. When insulin is bound to the insulin receptor, the serine / threonine residues of the forkhead O 3 (FOXO3) are phosphorylated by AKT. Phosphorylation of FOXO3 by AKT blocks entry into the nucleus and thereby prevents the expression of the forkhead O 3 (FOXO3) dependent subgenus. Conversely, in the case of growth factor subtype or cell stress conditions, the forkhead box O 3 (FOXO3) is dephosphorylated and enters the nucleus to express the subgenus (Martins R, et al., Aging Cell, 196-207, 2016).

(FOXO3) has been implicated in breast cancer (Lin H, et al., PLoS One, 2010), melanoma (Segura MF, et al., Proc Natl Acad Sci USA, 1814-1819, 2009) Anticancer Drugs., 898-907, 2014), kidney cancers (Lee J et al., Biosci, et al., Int J Oncol., 1613-20, 2009), lung cancer (Liu H. et al. Biotechnol Biochem, 1947-53, 2016), and it acts as a tumor suppressor in cancer (Coomans de Brachene A., Cell Mol Life Sci., 1159-72, 2016).

O- GlcNAc glycation is a type of post translational modification (PTM) that acts on serine / threonine residues of intracellular proteins. This takes place by (O- GlcNAc transferase) OGT OGT the N on serine / threonine residues of proteins - the action of a sugar chain attached acetylglucosamine (N -acetylglucosamine). O- GlcNAc glycation can share the phosphorylation point of intracellular proteins and act competitively. (Hart GW, et al., Nature, 1017-22, 2007), but it has been reported that the O- ring of the forkhead box O 3 (FOXO 3) has an effect on the signal transduction, protein expression, protein degradation, GlcNAc glycation and the relationship with malignant cancer are not known.

In addition to surgery, radiotherapy and chemotherapy are performed to treat cancer, but most of them have limited effect on the survival period of the patients. In the case of drugs called therapeutic agents, the efficacy can only be confirmed through studies on people who have clinical studies even after various stages of research. Therefore, it is very important to develop an analytical system capable of measuring the degree of efficacy of a cancer treatment agent.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a method for screening anticancer substances, predicting therapeutic prognosis, and determining the susceptibility of an individual to an anticancer drug, by measuring the level of O- GlcNAc glycosylation of FOXO3 protein .

The present invention also provides a composition capable of screening an anticancer substance comprising an antibody specifically binding to the O- glycNAc glycation site of a fork head box O 3 (FOXO3) protein and a composition capable of screening for an O- -GlcNAc < / RTI > saccharification-inhibiting substance.

The present invention provides a method for analyzing the level of FOXO3 protein comprising examining whether O- GlcNAc glycosylation of a forkhead O 3 (FOXO3) protein.

In addition, the present invention provides a method for screening anticancer substances comprising measuring the O- glycNAc glycosylation level of a fork hair box O 3 (FOXO3) protein.

The present invention also provides a composition for screening an anti-cancer agent comprising an antibody that specifically binds to O- GlcNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

The present invention also provides a method for predicting a therapeutic prognosis comprising measuring the level of O- GlcNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

In addition, the present invention provides a method for confirming susceptibility to an anticancer agent of an individual comprising measuring the level of O- GlcNAc glycation of a sample of forkhead box O 3 (FOXO3) protein.

In addition, the present invention provides an anticancer composition comprising a substance that inhibits O- glycNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

The present invention also provides a method for providing information for diagnosis of pancreatic cancer, comprising measuring the O- glycNAc glycosylation of a forkhead box O 3 (FOXO3) protein.

The present invention also provides a composition for cancer diagnosis comprising an antibody specifically recognizing O- GlcNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

The present invention also provides a cancer diagnostic kit comprising an antibody specifically recognizing O- GlcNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

In the present invention, the O- glycNAc glycosylation level of the FOXO3 (FOXO3) protein has been newly found to be an index and target of treatment in cancer, particularly malignant cancer including liver cancer, stomach cancer and pancreatic cancer, According to the present invention, the level of glycation can be measured to predict the sensitivity to an anticancer agent, the prognosis of treatment, and further screen for a substance having anticancer activity. In addition, a substance capable of specifically recognizing the O- glycNAc glycosylation of the fork hair box O 3 (FOXO3) protein or capable of specifically inhibiting the glycation can be used as an anticancer therapy prognostic prediction or anticancer agent.

FIG. 1A is a graph showing the result of analysis of O- GlcNAc level of whole protein by Western blotting by separating non-tumor region (N: tumor region) and tumor region (T: Tumor region) in pancreatic cancer patients .
FIG. 1B shows the results of H-score evaluation of O- GlcNAc glycation level in a part of pancreatic tissue by FOXO3 immunohistochemical staining and tissue of pancreatic cancer patient.
FIG. 2A shows the results of immunoassay using an anti-FOXO3 antibody after separating the non-tumor area (N) and the tumor stomach (T) from pancreatic cancer patients in pancreatic cancer patients. And the O- GlcNAc glycosylation level of the FOXO3 protein were analyzed by Western blotting.
2B is a flowchart illustrating a protein process according to an embodiment of the present invention. Figure 3a shows Western blot analysis of O- GlcNAc glycation levels in tissues of each patient with pancreatic cancer (n = 12).
FIG. 3B shows the results of immunohistochemistry and western blot analysis of the expression of fork hair box O 3 (FOXO3) protein and the level of O- GlcNAc glycation in various tissues (n = 12).
Figures 4a and 4b show the results of analysis of the level of forkhead O 3 (FOXO3) O- GlcNAc glycation in pancreatic cancer cell lines (HPDE, PANC-1, BxPC-3, HPAC).
FIG. 5 is a flowchart illustrating a process of forming a FOX-OV cell line according to an embodiment of the present invention.
6A is a flow chart illustrating a process of purifying FOXO3 from FOX-OV cells through pH fractionation and immunoprecipitation according to an embodiment of the present invention.
FIG. 6B shows the result of Western blot analysis of the cells expressing FOXO3-overexpressing FOXO3 cells using the PANC-1 cell line.
FIG. 6C shows the results of immunoprecipitation using an anti-FOXO3 antibody in a fox hair cell O 3 (FOXO3) overexpressing cell line followed by Western blotting of the separation of the fork hair box O 3 (FOXO3) protein using an OFFGEL classifier Show the results.
Figure 6d shows a schematic diagram for the O-linked glycosylation point of the forkhead box O3 (FOXO3).
FIG. 7a shows the result of Western blot analysis of the cells obtained by overexpressing glycoprotein in each of the fork hair box O 3 (FOXO3) glycosylation sites using the PANC-1 cell line.
FIG. 7B shows the results of qRT-PCR measurement of the expression of the fork hair box O 3 (FOXO3) target genes ( FasL , MnSod , Nib , p27 and p21 ) in the glycosylation overexpressing cells at each glycosylation site.
FIG. 7c shows the results of measuring the expression level of p21 in a cell line containing a glycosylation mutation (FOX-S284A) or a phosphorylation mutation (FOX-S284D) at S284 point of a fork head box O 3 (FOXO3).
FIG. 8A shows the results of measuring the number of whole cells according to time-series after post-transcription modification of the S284 site of the fork head box O 3 (FOXO3) in the pancreatic cancer cell line (PANC-1).
FIG. 8B is a graph showing the proliferation rate of cells according to WST-1 analysis according to the post-transcriptional transformation of the S284 site of the fork head box O 3 (FOXO3) in the pancreatic cancer cell line (PANC-1).
FIG. 8C shows the results of fluorescence analysis at 570 nm after dyeing and the change in the number of whole cells according to the post translational translation of the S284 site of the fork head box O 3 (FOXO3).
Figures 9a and 9b show the results of FACS cell cycle analysis on FOXO3 variants (FOX-OV, FOX-S284A or FOX-S284D).
FIG. 10a is a graph showing the effect of the anticancer drugs (gemcitabine (Gem), doxorubicin (Dox), actinomycin di (Act D), paclitaxel (Pac), salinomycin (Sal) and BG (benzyl-2- -deoxy- [alpha] -D-galactopyranoside, OGT inhibitor)) was observed at the IC ₅₀ value of the drug: Gem, 92 μM; Dox, 68.5 [mu] M; ActD, 380 nM; Pac, 104 [mu] M; And Sal, 830 nM.
Figure 10b, 10c and 10d are then immunoprecipitated the fork head box O 3 (FOXO3) protein in pancreatic cancer cells treated with pancreatic cancer therapeutic agent, wherein - with the O -GlcNAc antibody FOXO3, p21, fork head box 3 O (FOXO3 ) Protein by Western blotting of the O- GlcNAc glycation level of the protein.

The present invention relates to a method for screening anticancer substances, estimating the prognosis of treatment, confirming the susceptibility of an individual to an anticancer drug, and providing information for cancer diagnosis by measuring the O- GlcNAc glycation level of a fork hair box O 3 (FOXO3) protein.

The present invention also provides a composition capable of screening an anticancer substance comprising an antibody specifically binding to the O- glycNAc glycation site of a fork head box O 3 (FOXO3) protein and a composition capable of screening for an O- -GlcNAc < / RTI > glycation, and compositions and kits for cancer diagnosis.

Hereinafter, the present invention will be described in detail.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, unless further departing from the scope of the invention as defined by the following claims. It is not. It is to be understood that the scope of the present invention includes all modifications, equivalents, and alternatives falling within the scope and spirit of the present invention.

The present invention has confirmed that it is possible to estimate the anticancer activity evaluation, anticancer drug sensitivity, and prognosis of chemotherapy using the increase or decrease of the O- GlcNAc glycation level of the Forkhead O 3 (FOXO3) protein. The invention relates to a method for analyzing the level of FOXO3 protein comprising examining whether O- GlcNAc glycosylation of a forkhead box O3 (FOXO3) protein.

Whether or not the O- GlcNAc glycosylation can be examined using a known technique, specifically, can be performed using an antibody specifically recognizing O- GlcNAc glycation.

According to one embodiment of the present invention, an anti- O- GlcNAc (RL2): ab2739 antibody specifically recognizing anti-FOXO3: 75D8, D19A7 (Cell signaling technology) and O- (abcam), FOXO3 protein expression and O- glycNAc glycosylation were confirmed. As a result, expression of FOXO3 protein was high in tumor tissues of pancreatic cancer patients and O- GlcNAc glycation was detected.

In the present invention, the Forkhead box O 3 (FOXO3) protein is a transcription factor that acts at the lowest level of the insulin-like growth factor signaling pathway. The inventors of the present invention have found that FOXO3 protein is overexpressed in solid cancer cells such as pancreatic cancer, stomach cancer, and liver cancer. As a result of repeated studies on FOXO3 protein as a therapeutic target, O- GlcNAc glycation of FOXO3 protein occurs in cancer cells The present invention has been accomplished based on the findings that it is possible to predict the treatment of cancer through the increase or decrease of the glycation level, screen the anticancer agent, and further kill the cancer cells.

In one embodiment of the present invention, it has been confirmed that the O- GlcNAc glycosylation of the FOXO3 protein occurs only in the cancer cells. The present invention can estimate the therapeutic effect of the cancer by evaluating the presence or absence of O- GlcNAc glycosylation of the FOXO3 protein have.

The Fork Hair Box O 3 (FOXO3) protein according to the present invention may be composed of the amino acid sequence of SEQ ID NO: 1.

[SEQ ID NO: 1]

MAEAPASPAPLSPLEVELDPEFEPQSRPRSCTWPLQRPELQASPAKPSGETAADSMIPEEEDDEDDEDGGGRAGSAMAIGGGGGSGTLGSGLLLEDSARVLAPGGQDPGSGPATAAGGLSGGTQALLQPQQPLPPPQPGAAGGSGQPRKCSSRRNAWGNLSYADLITRAIESSPDKRLTLSQIYEWMVRCVPYFKDKGDSNSSAGWKNSIRHNLSLHSRFMRVQNEGTGKSSWWIINPDGGKSGKAPRRRAVSMDNSNKYTKSRGRAAKKKAALQTAPESADDSPSQLSKWPGSPTSRSSDELDAWTDFRSRTNSNASTVSGRLSPIMASTELDEVQDDDAPLSPMLYSSSASLSPSVSKPCTVELPRLTDMAGTMNLNDGLTENLMDDLLDNITLPPSQPSPTGGLMQRSSSFPYTTKGSGLGSPTSSFNSTVFGPSSLNSLRQSPMQTIQENKPATFSSMSHYGNQTLQDLLTSDSLSHSDVMMTQSDPLMSQASTAVSAQNSRRNVMLRNDPMMSFAAQPNQGSLVNQNLLHHQHQTQGALGGSRALSNSVSNMGLSESSSLGSAKHQQQSPVSQSMQTLSDSLSGSSLYSTSANLPVMGHEKFPSDLDLDMFNGSLECDMESIIRSELMDADGLDFNFDSLISTQNVVGLNVGNFTGAKQASSQSWVPG

The site where glycosylation occurs in the FOXO3 protein may be any site selected from the group consisting of S284, S286, S411, T475, S476, S482, S551, and combinations thereof in the amino acid sequence of SEQ ID NO: The glycation may occur at the site at the same time, or may occur only at a part or at any one site. Therefore, screening for anticancer substances, prediction of prognosis of chemotherapy, or susceptibility to anticancer drugs can be confirmed by evaluating the presence or absence of glycation occurring at the site.

In particular, when the S284 region of the FOXO3 protein is not glycosylated, it is confirmed that the growth of cancer cells is impossible, and it is preferable to evaluate the glycosylation and glycosylation of the S284 region.

In the present invention, "an individual" may be a patient suspected of having a tumor, or a patient whose diagnosis of cancer has been confirmed.

In the present invention, the term "cancer" includes both solid cancer and blood cancer. Specific examples thereof include, but are not limited to, gastric cancer, colon cancer, liver cancer, pancreatic cancer, breast cancer, lung cancer and cholangiocarcinoma . In the present invention, it may preferably be a digestive cancer including liver cancer, pancreatic cancer, stomach cancer and the like.

The term "anticancer substance" used in the present invention can be used in combination with an "anticancer agent ", which is a substance capable of killing or reducing cancer cells or inhibiting the growth of cancer cells and includes compounds, nucleic acid molecules, It means.

The term "sample " in the present invention means a substance or organism which is provided for testing, inspection or analysis for a specific purpose. Specifically, in the present invention, a tissue, a cell, a blood And an animal other than a human being who has been manipulated to have the cancer for the screening of anticancer substances in the present invention. The sample may be a cell expressing a forkhead box O 3 (FOXO3) protein or a forkhead box O 3 (FOXO3) protein.

In the present invention, "tumor cell" means a cancer cell which grows indefinitely in normal tissue cells and continues to grow rapidly regardless of tissue state or environment, and includes all cells having the characteristics of typical cancer cells. May be obtained from tumor tissue isolated from an individual, or may be a commercially available cancer cell line, and may be used in the present invention in the form contained in the sample.

In the present invention, it may preferably be a cancer cell strain that expresses a forkhead box O 3 (FOXO3) protein. (Pancreatic cancer cell line), HPAC (pancreatic cancer cell line), BxPC-3 (pancreatic cancer cell line), AGS (gastric cancer cell line) and Hep3B2.1-7 (hepatocellular carcinoma cell line), hepatocellular carcinoma cell line ), But is not limited thereto.

In the present invention, " level of protein analysis ", "measurement of protein expression level ", or" measurement of glycation level of protein "is used to determine the presence and expression level of a protein expressed in a cancer marker gene in a biological sample As an example, the amount of protein can be determined using an antibody that specifically binds to the protein of the gene. Immunoprecipitation assay, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), tissue immunoassay, radioimmunodiffusion (ELISA), radioimmunoassay, , Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, FACS and protein chip, but are not limited thereto.

In one embodiment of the present invention, a method for measuring the efficacy of a therapeutic agent for pancreatic cancer treated with a pancreatic cancer cell line comprises concentrating the protein using an antibody that specifically binds to the fox hair cell O 3 (FOXO3) protein, by using an antibody specifically binding to GlcNAc O- confirmed whether the change of O- GlcNAc glycosylation.

The method of screening for an anticancer material according to the present invention comprises: contacting a candidate material with a sample containing cells expressing a forkhead box O 3 (FOXO3) protein; Measuring the O- GlcNAc glycosylation level of the forkhead box O 3 (FOXO3) protein of the sample; And when the O- GlcNAc glycation level of the sample after contact with the candidate substance is lower than the O- GlcNAc glycation level of the FOXO3 protein of the sample before contact with the candidate substance, the candidate substance is judged to have anticancer activity Can be included. The O- glycNAc glycosylation of the FOXO3 protein of the present invention has higher accuracy than that of expression at the protein level and at the gene level. In particular, even if a change in the expression level of the gene is not detected, the change in the expression level can be detected at the protein level, and the anticancer activity of the substance can be confirmed with higher sensitivity.

In one aspect of the present invention, the present invention relates to a composition for screening an anticancer substance or a kit for screening an anticancer substance, which comprises an antibody specifically recognizing an O- glycNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

In the present invention, "specifically binding" or "specifically recognizing" means that the binding or recognizing ability to a target substance is superior to other substances, or only the target substance is reacted ≪ / RTI >

"Antibody" in the present invention refers to an immunoglobulin gene or immunoglobulin gene capable of specifically binding to an antigen or an epitope, or a peptide or polypeptide substantially encoded thereby derived from or fragments thereof. Antibodies in the present application include various forms of antibody structures including, but not limited to, whole antibodies and antibody fragments. The antibody fragment comprises a portion of the full length antibody, the variable domain of the antibody, or at least the antigen binding site of the antibody. Examples of antibody fragments include multispecific antibodies formed from diabodies, single-chain antibody molecules and antibody fragments.

In one embodiment, the antibody may be a polyclonal antibody or a monoclonal antibody. Antibodies to forkheadbox O 3 (FOXO3) protein and O- GlcNAc can be generated using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 ), Recombinant DNA methods (US Patent No. 4,816,56) or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol. , ≪ / RTI > 1-597 (1991)). General procedures for antibody preparation are described in Harlow E. and Lane D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art . The polyclonal antibody can be obtained by injecting a suitable animal with a forkhead box O 3 (FOXO3) protein antigen, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique have.

The kit may include an antibody specific for a fox headbox O 3 (FOXO3) protein and an antibody specific for O- GlcNAc glycosylation, respectively.

For example, the kit can be used to detect and quantify a target protein using an antibody against the FOXO3 protein, and then separate and detect the glycated protein using an antibody specific for O- GlcNAc. Can be performed.

The composition or kit may further comprise additional components that are typically included for detection of the subject substance. For example, distilled water or a buffer solution, but the kind thereof is not limited as long as it is included in a conventional composition and kit of the present invention.

In another aspect of the present invention, the present invention relates to a method for predicting a therapeutic prognosis comprising measuring the O- GlcNAc glycosylation level of a fork hair box O 3 (FOXO3) protein of a sample obtained from an individual .

In the case of O- glycNAc glycosylation of the fox hair-box O 3 (FOXO3) protein, the glycation is detected only in the tumor site in the tumor site and the non-tumor site in the tissue of the individual. In the case where the O -GlcNAc glycosylation does not occur, Growth has been inhibited through one embodiment of the present invention.

Therefore, the O- glycNAc glycation level of the FOXO3 protein and the O- GlcNAc glycation level of the FOXO3 protein in the sample isolated from the individual after the chemotherapy treatment in the sample isolated from the individual before the chemotherapy treatment were compared with the O If the glycNAc glycation level is lower, it can be judged to have a therapeutic effect. In addition, such prognostic prediction can be used for conventional chemotherapeutic treatment methods such as chemotherapy using chemotherapeutic agents, radiation therapy and the like.

In another aspect of the present invention, the present invention provides a method for treating cancer, comprising: treating a sample obtained from an individual with an anticancer agent; And measuring the O- GlcNAc glycosylation level of the fork hair box O 3 (FOXO3) protein in the anticancer agent-treated sample, to a method for confirming susceptibility to an anticancer agent.

The method if compared to the O -GlcNAc glycosylation levels of cancer-treated FOXO3 O -GlcNAc glycation level and the anticancer treatment after FOXO3 protein of the protein in the sample, after anticancer treatment is O -GlcNAc glycosylated protein level of FOXO3 lower, cancer And judging that there is a susceptibility to < RTI ID = 0.0 >

When the present invention is used, the susceptibility of each individual to an anticancer agent can be confirmed before the administration of the anticancer agent, thereby increasing the therapeutic effect. In particular, since the sensitivity can be confirmed at the protein level, more precise measurement can be performed It is possible to select an anticancer agent highly sensitive to an anticancer agent, and ultimately, the effect on the treatment of cancer can be remarkably improved.

In another aspect, the present invention relates to an anticancer composition comprising a substance that inhibits O- GlcNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

The substance inhibiting the O- glycNAc glycosylation of the fork hair box O 3 (FOXO3) protein is not limited to the kind of compound, antibody, nucleic acid molecule and the like, as long as it can inhibit O- GlcNAc glycosylation. In the present invention, it has been confirmed that the growth of cancer cells does not normally occur when O- glycNAc glycation of the fork hair box O 3 (FOXO3) protein is inhibited. As a result, O- GlcNAc glycation Can be an important target in chemotherapy.

Preferably, the substance is selected from the group consisting of S284, S286, S411, T475, S476, S482, S551, and combinations thereof in the amino acid sequence of SEQ ID NO: O- GlcNAc < / RTI > glycation, preferably inhibiting the O- GlcNAc glycosylation at the S284 site in the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, when glycosylation at the S284 site does not occur, it is confirmed that cancer cell growth does not occur. As a result, it is possible to obtain an excellent anticancer activity by inhibiting glycosylation at S284.

The O- GlcNAc glycosylation inhibiting substance is a substance having functionality as an anticancer agent, and may be a nucleic acid, a protein, a natural product, a natural-derived extract or a compound.

In another aspect of the present invention, the present invention provides a method for diagnosing Pancreatic Cancer comprising measuring the O- glycNAc glycosylation of a fork head box O 3 (FOXO3) protein of a tumor sample of a tissue isolated from an individual The present invention relates to a method of providing information. In the case of the fork hair box O 3 (FOXO3) protein, it was confirmed in one embodiment of the present invention that the glycation is detected only in the tumor site in the tumor site and the non-tumor site in one tissue. Therefore, it is possible to diagnose pancreatic cancer when the glycation occurs or the glycation level is higher than that of the control group, as a result of measuring the O- glycNAc glycosylation of the fork hair box O 3 (FOXO3) protein in the sample isolated from the subject .

The control may be a healthy individual (normal human) without pancreatic cancer, or may be a non-tumor tissue of a subject expected to have pancreatic cancer. In particular, the O- glycNAc glycosylation of the fork hair box O 3 (FOXO3) protein of the present invention shows marked differences in the glycosylation sites between the tumor site and the non-tumor site even in the same individual, And to provide information for diagnosis of pancreatic cancer.

Further, in another aspect of the present invention, the present invention relates to a composition for diagnosing cancer comprising an antibody specifically recognizing O- GlcNAc glycosylation of a fork hair box O 3 (FOXO3) protein.

The FOXO3 protein of the present invention is characterized by O- glycNAc glycosylation only at the tumor site. Thus, when an antibody specifically recognizing O- GlcNAc glycosylation of the FOXO3 protein is used, It is possible to detect the glycosylation of the FOXO3 protein for diagnosis of cancer.

The antibody may specifically recognize O- GlcNAc glycation at any one position selected from the group consisting of S284, S286, S411, T475, S476, S482, S551, and combinations thereof in the amino acid sequence of SEQ ID NO: have.

In order to avoid the description of overlapping with respect to the saccharification and the like of the antibody and the protein, the above-described methods are also applied to the cancer diagnostic composition.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Experimental Examples. The following Examples and Experiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

[ Example  1] in pancreatic cancer Fork head box  O 3 ( FOXO3 ) ≪ / RTI > O- GlcNAc  Glycation level analysis

[ Example  1-1] Patients with pancreatic cancer Non-tumor vs  In tumor tissue FOXO3 O- GlcNAc Glycation  Whether and to what level

In order to determine the level of O- GlcNAc glycation of whole proteins in the tumor site versus the non-tumor site of pancreatic cancer patient tissue, electrophoresis and immunoblotting were performed.

The tissue obtained was obtained from Severance Hospital Gene Bank and Digestive Medicine Internal Medicine and was followed by the Clinical Trials Commission (IRB) regulations. The tissue samples were kept at -70 ℃ until the experiment.

For immunoblotting and immunoprecipitation analysis, the tissues of 12 patients with pancreatic ductal adenocarcinoma (PDAC) were divided into a tumor site and a non-tumor site (a non-tumor site adjacent to a pancreatic cancer site), and a lysis buffer (150 mM chloride -Sodium, 50 mM Tris pH 7.4, 1% NP-40, sodium-deoxycholic acid, sodium-vanadate] was added to perform sonication. After precipitation at 14,000 rpm for 30 minutes, the supernatant was recovered and again settled at 14000 rpm for 20 minutes to recover the supernatant.

Western blots were performed using antibodies from the same amount of protein. After 20 μg of the protein was subjected to 10% SDS-PAGE analysis, the whole gel was transferred to a nitrocellulose (NC) membrane for Western blotting, and then washed with TBS-T buffer solution containing 5% skimmed milk [20 mM Tris, 137 mM Chloride-Sodium, 0.1% Tween-20, pH 7.6] for 1 hour. Anti- O- GlcNAc (RL2, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) The pear was subjected to secondary treatment. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

As a result, it was found that the level of O- GlcNAc glycation was increased only in the tumor area of pancreatic cancer patients (9.2 ± 0.52 times, n = 3), as shown in FIG.

In addition, immunohistochemical staining (IHC) was used to identify FOXO3 protein levels in both the tumor site and adjacent non-tumor site nuclei in a set of 16 tumor tissues of pancreatic cancer (PDAC) patients. All data were calculated as the mean of H-score and 16 case experiments.

As shown in Figs. 1B and 1C, it was confirmed that FOXO3 expression continued more steadily in the tumor site than in the non-tumorous site.

Immunoprecipitation, immunoblot analysis and electrophoresis were performed to analyze the presence and expression level of FOXO3 and the level of O- GlcNAc glycosylation of FOXO3 in pancreatic cancer tissues adjacent to the pancreatic cancer.

2 μg of anti-forkhead O 3 (FOXO3) antibody (75D8, Cell Signaling Technology) and 10 μl of protein G agarose were immunoprecipitated together in 1 ml tubes at 4 ° C. overnight using 1 mg of tissue lysate , Followed by 10% SDS-PAGE electrophoresis.

Western blotting was performed using the antibody. After 10% SDS-PAGE analysis, the entire gel was transferred to a nitrocellulose (NC) membrane for Western blotting, and then washed with TBS-T buffer solution containing 5% skimmed milk [20 mM Tris , 137 mM chloride-sodium, 0.1% Tween-20, pH 7.6] for 1 hour. The anti-FOXO3 (75D8, Cell Signaling Technology) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) 5000 times. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

In addition, immunoprecipitation analysis was carried out using ten tissues divided into a tumor site and a non-tumor site, and the result was subjected to Western blotting using an anti-FOXO3 antibody. After 10% SDS-PAGE analysis, the entire gel was transferred to a nitrocellulose (NC) membrane for Western blotting, and then washed with TBS-T buffer solution containing 5% skimmed milk [20 mM Tris , 137 mM chloride-sodium, 0.1% Tween-20, pH 7.6] for 1 hour. Anti- O- GlcNAc (RL2, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) The pear was subjected to secondary treatment. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

As a result, as shown in Figs. 2A and 2B, a clear band appeared in the Western blot, indicating that the increase in O- GlcNAc was due to FOXO3 protein binding. In addition, FOXO3 O- GlcNAc glycation levels were found to be higher (7.37 ± 1.68 times, n = 3) than tumor sites (T). However, the detection of O- GlcNAc glycosylated protein was low in non-tumorous areas of patients with pancreatic cancer.

The result of immunoprecipitation is subjected to 10% SDS-PAGE analysis and the protein is stained with Coomassie blue dye. After image analysis, mass spectrometry was performed on the bands to confirm the presence of FOXO3. Protein bands were picked from the gel stained with Coomassie blue dye and decolorized and digested with trypsin before the cleaved peptides were mixed with a mixture of Poros R2 and Oligo R3 resin ≪ / RTI >

(Q-TOF MS / MS) (Agilent) was used to further confirm that the bands detected by Western blotting using the anti- O- GlcNAc mAb were exactly identical to the FOXO3 protein 6530) was used to observe the ablated protein band in the gel. The spectra obtained with Q-TOF-MS were identified using the MASCOT database. The results are shown in Table 1 below.

number Protein name mass Score pl value Query smatch sequence
Coverage gi42519916 Forkhead box protein O3 71517 1455 4.99 227 77%

 As shown in Table 1, the protein cleaved from the gel was actually FOXO3 (MW 71517, sequence coverage: 77%).

[ Example  1-2] Except pancreas Other  Long-term FOXO3  Expression and O- GlcNAc Glycation  Level analysis

In addition, 12 pairs of tissue samples from individual PDAC patients were examined to confirm the reproducibility of the results, with all tumor sites showing higher O- glycNAc glycosylation (4.61 0.38 times, n = 12) in FOXO3.

Immunoprecipitation and immunoblotting were performed to analyze the level of O- GlcNAc glycosylation of FOXO3 in tissues of various cancer patients other than the pancreas. (10)), gastric carcinoma (10), hepatocellular carcinoma (10), cholangiocarcinoma (BD), colon cancer (10) and lung cancer (LC) were similar to those of pancreatic cancer (12) The patient's tissues were divided into the tumor site and the non-tumor site (non-tumor site adjacent to the tumor site), followed by immunoprecipitation analysis and Western blotting using the antibody. The resultant was subjected to 10% SDS-PAGE analysis, and then the entire gel was transferred to a nitrocellulose (NC) membrane for Western blotting. Then, TBS-T buffer solution containing 5% skimmed milk [ 20 mM Tris, 137 mM Chloride-Sodium, 0.1% Tween-20, pH 7.6] for 1 hour. Anti- O- GlcNAc (RL2, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) And then subjected to secondary treatment. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

As a result, as shown in FIGS. 3A and 3B, it was confirmed that O- GlcNAc glycosylation of FOXO3 is more expressed in the tissues of cancer patients of digestive system such as pancreatic cancer, liver cancer and stomach cancer. Specifically, more glycation occurred at the levels of gastric cancer (gastric cancer, 2.65 ± 0.31 fold, n = 10) and liver tumors (hepatocellular carcinoma, HAC) at 1.76 ± 0.46 fold, n = 10). The highest changes in FOXO3 O- GlcNAc glycation levels were found in pancreatic (PDAC) tumors (vs. GC tumors or HCC tumors).

[ Example  2] within pancreatic cancer cells Fork head box  O 3 ( FOXO3 ) Over-expressing cell line production

[Example 2-1] Production of overexpressing cell line of FOXO3 in pancreatic cancer cells

To identify the O- GlcNAc glycosylation site of FOXO3, we overexpressed the FOXO3 cell line in pancreatic cancer cell lines.

First, in several pancreatic cancer cell lines, FOXO3 O- GlcNAc To confirm the glycation level, HPDE, PANC- 1, BxPC- 3, and HPAC cell lines were treated with Western Blot and immunoprecipitation assays were performed. The results are shown in the relative O- GlcNAc glycation ratio relative to GAPDH (normalized control), and the results are shown in Figures 4a and 4b.

As shown in Figures 4a and 4b , the overall O- GlcNAc The level of glycation was significantly increased in the pancreatic cancer cell line ( PANC-1) compared to the unknown cell line HPDE. In addition, the level of FOXO3 was also increased in PANC-1, BxPC-3 and HAPC cell lines, and increased in PANC-1 and HAPC cell lines, which have a higher relative ratio of O- GlcNAc to FOXO3.

After confirming the presence of the glycosylation FOXO3 O -GlcNAc significantly enhanced in PDAC confirmed that (4.61 ± 0.38-fold, n = 12), specific O -GlcNAc glycosylation sites, and further to quantify the level of O -GlcNAc FOXO3 related to cancer-cell proliferation And tried to identify the potential role of these sites. To this end, FOXO3-overexpressing cell lines were prepared to quantify O- GlcNAc levels and map specific regions within FOXO3.

As shown in FIG. 5A, PANC-1 (human pancreas epithelioid carcinoma) was used as a pancreatic cancer cell line and overexpressed in cells using a pCMV6-FOXO3 :: GFP plasmid vector. (DMEM, Gibco) mixture (10% Fetal Bovine Serum, Gibco, 1% Penicillin-Streptomycin, Gibco) containing 2.2 X 10 ⁶ cells in a 100 mm Petri dish ]. Two days later, the cells were cultured for one day in Opti-MEM (Gibco) containing no serum component. The plasmid vector containing the forkhead box O 3 (FOXO3) and the lipofectamine (Lipofectamine 3000, Invitrogen) were mixed well and incubated at 37 ° C for 2 hours and then cultured again with Dulbecco's modified Eagle medium mixture. After stabilization for two days, cells not overexpressed were removed using G418 (Sigma). This cell line was named FOX-OV.

[ Example  3] Q- Exactive orbitrap  LC- MS  Using a mass spectrometer Fork head box  O 3 (FOXO 3) O- Analysis of GlcNAc glycation point

 [ Example  3-1] Fork head box  O 3 ( FOXO3 ) Over-expressing cell line Fork head box  Isolation of O 3 (FOXO3) Protein

To obtain the purified FOXO3 protein for O- GlcNAc glycation point analysis by MS, immunoprecipitation assays, electrophoresis, OFFGEL classification and Western blotting were performed.

Protein was classified by pH using OFFGEL classifier (OFFGEL Fractionator 3100, Agilent) using 10 mg of cell lysate. Add rehydrated solution [IPG (pH 3-10, Agilent) solution, distilled water] to each column of the machine, then add cell lysate. Mineral oil was used to reduce the loss and voltage was applied from 800V to 8000V (FIG. 6A).

The proteins classified in the classifier were subjected to western blotting using an anti-FOXO3 antibody. After 10% SDS-PAGE analysis, the whole gel was transferred to a nitrocellulose (NC) membrane for Western blotting, and then a TBS-T buffer solution containing 5% skimmed milk [20 mM Tris , 137 mM chloride-sodium, 0.1% Tween-20, pH 7.6] for 1 hour. Anti- O- GlcNAc (RL2, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) The pear was subjected to secondary treatment. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

As a result, as shown in FIG. 6B, the protein was detected only in the FOXO3-overexpressing cell line prepared in Example 2, and it was not detected in the PANC-1, Mock and GFP cell lines. In addition, glycation was also detected only in the FOXO3-overexpressing cell line.

In addition, as shown in Fig. 6C, the concentrated protein was theoretically found to be a FOXO3 protein with a pH of 5.24, and the results showed that the recombinant FOXO3-GFP protein overexpressed in the FOXO3 over-expressing cell line (FOX-OV) Is equal to the expected size.

[Example 3-2] Analysis of glycation point of fork head box O 3 (FOXO 3)

OFFGEL products were immunoprecipitated overnight at 4 in a 1 ml tube together with 2 μg of anti-forkhead O 3 (FOXO3) antibody (75D8, Cell Signaling Technology) and 10 μl of protein G agarose, followed by 10% SDS- PAGE electrophoresis. The separated immunoprecipitated product is stained with Coomassie blue dye. After image analysis, mass spectrometry was performed on the bands to confirm the presence of FOXO3. Protein bands were picked from gels stained with Coomassie blue dye and decolorized and digestion with endoprotease (combination of trypsin, Lys-C, Asp-N) followed by digestion of the cleaved peptides with Poros ) R2 and an Oligo R3 resin mixture. The protein identity was analyzed by Q-Exactive orbitrap LC-MS (Thermo Scientific), and the spectrum obtained by Q-Exactive orbitrap LC-MS was identified using MASCOT database. Thirteen independent experiments were performed after endo-protease treatment.

number Protein name mass Score pl value sequence
Coverage O- GlcNAc mutation site gi42519916 Forkhead box protein O3 71.2 10969 ± 4691 5.16 92.72% S284, S286, S411, T475, S476, S482, S551

protein location Score Charge MH + [Da] DELTA M [ppm] Miss Sequence


FOX03


S284 11.55 3 2222.01 -5.16 One DDsPSQLSKWPGSPTSRSS S286 14.31 3 2579.15 -9.13 2 DDSPsQLSKWPGSPTSRSSDEL S411 37.5 2 1220.56 -3.09 0 sSSFPYTTK T475 16.16 2 1377.64 0.41 One DLLtSDSLSHS T475 / S476 18.34 2 1580.71 -3.75 One DLLtsDSLSHS S482 23.69 2 1377.64 -1.98 0 DLLTSDSLSHs S551 100.09 2 2229.05 -0.10 0 ALsNSVSNMGLSESSSLGSAK

As a result, seven points (S284, S286, S411, T475, S476, S482 and S551) of glycosylation sites of FOXO3 were newly found, as shown in Tables 2, 3 and 6D. All glycosylation sites of FOXO3 were normally located on the transactivation domain (TAD) where the transcriptional regulator adheres. Repeated, stringent MS analysis showed that the mean score of recombinant overexpressed FOXO3 was 10969.46 ± 4691 (n = 13).

[Example 4] Functional analysis of the glycation point of fork head box O 3 (FOXO 3)

[ Example  4-1] Fork head box  O 3 ( FOXO3 )of Glycation By branch Glycation  Invisible plasmid vector production

O- GlcNAc glycosylation of FOXO3 To confirm the function of glycation at each site, overexpressing cell lines which can not be glycosylated at each glycosylation site of FOXO3 in pancreatic cancer cell line were prepared. The pCMV6-FOXO3 :: GFP plasmid vector used in the previously prepared fork hair box O 3 over-expressing cell line was subjected to site directed point mutagenesis using serine and threonine at the corresponding site Alanine (alanine) to produce a plasmid vector in which glycation did not occur. The primers used for the production of the non-glycosylated vector for each glycosylation site are shown in Table 4 below.

No Site Primer sequences One S284A (Sense) 5'-cgaatcagctgacgac GCT cccttcccagctctccaag-3 ' (Antisense) Gt; 2 S284D (Sense) 5'-cgaatcagctgacgac GAT cccttcccagctctccaag-3 ' (Antisense) 5'-cttggagagctgggaggg ATC gtcgtcagctgattcg-3 ' 3 S286A (Sense) 5'-cagctgacgacagtccc GCC cagctctccaagtggc-3 ' (Antisense) 5'-gccacttggagagctggg CGG gactgtcgtcagctg-3 ' 4 S411A (Sense) 5'-gggactcatgcagcgg GCC tctagcttcccgtatac-3 ' (Antisense) Gt; 5 T475A (Sense) Gt; (Antisense) 5'-ggctaagtgagtccga AGC gagcaggtcctggagtg-3 ' 6 S476A (Sense) 5'-ccaggacctgctcact GCG gactcacttagccacag-3 ' (Antisense) 5'-ctgtggctaagtgagtc CGC agtgagcaggtcctgg-3 ' 7 S482A (Sense) 5'-ggactcacttagccac GCC gatgtcatgatgacac-3 ' (Antisense) Gt; 8 S551A (Sense) 5'-gtggcagccgtgccttg GCG aattctgtcagcaac-3 ' (Antisense) Gt;

[ Example  4-2] Fork head box  O 3 ( FOXO3 )of Glycation By branch Glycation  Overexpression cell line production

To confirm the function of O- GlcNAc glycosylation site of FOXO3, a glycosylation-overexpressing cell line of FOXO3 at each glycation site was prepared in pancreatic cancer cell line. Using the plasmid vector prepared in Example 4-1, PANC-1 (human pancreas epithelioid carcinoma), which is a pancreatic cancer cell line, was overexpressed in each cell. (DMEM, Gibco) mixture (10% Fetal Bovine Serum, Gibco, 1% Penicillin-Streptomycin, Gibco) containing 2.2 X 10 ⁶ cells in a 100 mm Petri dish ]. Two days later, the cells were cultured for one day in Opti-MEM (Gibco) containing no serum component. Plasmid vector containing fork head box O 3 (FOXO3) and lipofectamine (Lipofectamine 3000, Invitrogen) are mixed well and incubated at 37 ° C for 2 hours, and then cultured in Dulbecco's modified Eagle medium mixture. After stabilization for two days, cells not overexpressed were removed using G418 (Sigma), and glycosylated cells were obtained at each site. The nomenclature for each cell line is shown as FOX-S284A, FOX-S286A, FOX-S411A, FOX-T475A, FOX-S476A, FOX-S482A and FOX-S551A, FOX-OV was a wild-type FOXO3 protein overexpressing cell line, and a Mock transformed with a vector not containing FOXO3 was used as a control.

No Cell name Description Remarks One FOX-OV FOXO3 overexpressing cell line
(FOXO3 overexpressed cell line) Wild type FOXO3
(Wild type FOXO3) 2 FOX-S284A S284 of FOXO3 is replaced with A
(S284 within FOXO3 was substituted to A) PTMs defective FOXO3
( O- GlcNAcylation and phosphorylation) 3 FOX-S286A S286 of FOXO3 is replaced with A
(S286 within FOXO3 was substituted to A) 4 FOX-S411A S411 of FOXO3 is replaced with A
(S411 within FOXO3 was substituted to A) 5 FOX-T475A T475 of FOXO3 is replaced with A
(T475 within FOXO3 was substituted to A) 6 FOX-S476A S476 of FOXO3 is replaced with A
(S476 within FOXO3 was substituted to A) 7 FOX-S482A S482 of FOXO3 is replaced with A
(S482 within FOXO3 was substituted to A) 8 FOX-S551A S551 of FOXO3 is replaced with A
(S551 within FOXO3 was substituted to A) 9 FOX-S284D S284 of FOXO3 is replaced with D
(S284 within FOXO3 was substituted to D) Phospho-mimeted FOXO3

Western blotting was performed using the antibody from the same amount of protein in order to confirm whether FOXO3 overexpression occurred well from the cell line prepared above. After 20 μg of the protein was subjected to 10% SDS-PAGE analysis, the whole gel was transferred to a nitrocellulose (NC) membrane for Western blotting, and then washed with TBS-T buffer solution containing 5% skimmed milk [20 mM Tris, 137 mM Chloride-Sodium, 0.1% Tween-20, pH 7.6] for 1 hour. Anti- O- GlcNAc (RL2, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) The pear was subjected to secondary treatment. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

As a result, as shown in FIG. 7A, it was confirmed that overexpression of FOXO3 was well performed in all the overglycosylated cell lines of the fork hair box O 3 (FOXO3) at each glycation point.

[ Example  4-3] Fork head box  O 3 ( FOXO3 )of Glycation  Identify branch office features

The glycation points of the forkhead box O 3 (FOXO3) identified above were all located on the transfer facilitating point (TAD). This is a point related to the regulation of the expression of the sub-genes in association with FOXO3. Therefore, we investigated whether O- GlcNAc glycation affects the expression of various FOXO3 target genes at each site. To confirm the function of the glycation site, the subgenus of FOXO3 ( FasL, MnSod, Nib, p27 and p21 ) Respectively.

Each of the over-expressing cell lines prepared in Example 4-2 was collected, RNA was isolated using Trizol, and RNA was purified using an RNeasy kit. After that, cDNA was synthesized using reverse transcriptase. Real-time quantitative real-time PCR amplification was performed using the CFX Real-Time PCR detection system using the corresponding primers of each gene (Table 5), 10 mM dNTP, and SYBR green PCR mix. Expression of the gene was analyzed.

No Gene Primer sequences One FOXO3 (Forward) 5'-CAACCAGCTCCTTTAACAGC-3 ' (Reverse) 5'-TAAGTGAGTCCGAAGTGAGC-3 ' 2 FasL (Forward) 5'-TCCGTGAGTTCACCAACCAAA-3 ' (Reverse) 5'-GGGGGTTCCCTGTTAAATGGG-3 ' 3 MnSod (Forward) 5'-CAGACCTGCCTTACGACTATGG-3 ' (Reverse) 5'-CTCGGTGGCGTTGAGATTGTT-3 ' 4 Nib3 (Forward) 5'-CTGGGTAGAACTGCACTTCAG-3 ' (Reverse) 5'-GGAGCTACTTCGTCCAGATTCAT-3 ' 5 p21 (Forward) 5'-CCTGGTGATGTCCGACCTG-3 ' (Reverse) 5'-CCATGAGCGCATCGCAATC-3 ' 6 p27 (Forward) 5'-TCTCTTCGGCCCGGTCAAT-3 ' (Reverse) 5'-AAATTCCACTTGCGCTGACTC-3 ' 7 GAPDH (Forward) 5'-ATCAAGAAGGTGGTGAAGCA-3 ' (Reverse) 5'-ACCAGGAAATGAGCTTGACA-3 '

As a result, as shown in FIG. 7B, most of the expression of the FOXO3 target gene did not change, but only the p21 gene (cyclin dependent kinase inhibitor 1, CDKN1A) of the subgenus increased its expression amount when there was no glycosylation at S284 of FOXO3 (57.6 ± 2.91%, p = 0.0015, relative value of FOX-S284A mutant cells to FOX-OV cells).

 [ Example  4-4] Fork head box  O 3 ( FOXO3 )of Saccharomyces and  Identify differences in phosphorylation

In order to eliminate the effect of S284 phosphorylation on the expression of p21 in FOXO3, the pCMV6-FOXO3 :: GFP plasmid vector was transfected with serine and threonine at the corresponding site using the site directed point mutagenesis method (phospho-mimic) plasmid vector was prepared using the primers shown in Table 3, replacing threonine with aspartic acid.

In addition, the human pancreatic epithelioid carcinoma (PANC-1), which is a pancreatic cancer cell line, was overexpressed using the previously prepared phosphorylated mimic plasmid vector. (DMEM, Gibco) mixture [10% Fetal Bovine Serum (Gibco), 1% Penicillin-Streptomycin, Gibco) containing 2.2 x 10 ⁶ cells in a 100 mm Petri dish ]. Two days later, the cells were cultured for one day in Opti-MEM (Gibco) containing no serum component. The plasmid vector containing the forkhead box O 3 (FOXO3) and lipofectamine (Lipofectamine 3000, Invitrogen) were mixed well and incubated at 37 ° C for 2 hours and then cultured in Dulbecco's modified Eagle's medium mixture. After stabilization for two days, cells not overexpressing were removed using G418 (Sigma). The over-expressing cell line produced was named FOX-S284D (see Table 3) (with phosphate-mimic FOX-OV to distinguish the loss effect of O- GlcNAc glycation from S284 phosphorylation). RNA was isolated using Trizol, and RNA was purified using an RNeasy kit. After that, cDNA was synthesized using reverse transcriptase. Real-time quantitative real-time PCR was performed using the corresponding primers of each gene, 10 mM dNTP and SYBR green PCR mix, and the expression of the sub-genes was detected using the CFX Real-Time PCR detection system Respectively.

The function of O- GlcNAc glycosylation of FOXO3 was confirmed in the pancreatic cancer cell line. The over-glycosylation-overexpressing cell line of FOXO3 was prepared and used in the same manner as in Example 4-2. In addition, the expression of p21 gene was analyzed using Mock and previously prepared FOX-OV.

As a result, as shown in Figure 7c for, when in the cell line FOX-S284A to S284 FOXO3 site of glycosylation is not in progress if it does not express (S284 Lacking O -GlcNAc) and FOX-S284D (when not phosphorylated) of only p21 It was confirmed that the expression level was increased. This means that O- GlcNAc glycation, rather than phosphorylation, in FOXO3 S284 inhibits the expression of p21 , indicating loss of FOXO3 tumor suppressor activity.

[Example 4-5] p21 Functional regulation of cell function

In fact, p21 is a cell cycle-dependent kinase inhibitor ( p21 ) that is involved in cell cycle and cell proliferation. Therefore, it was confirmed that the restored p21 protein could inhibit the activity of the cyclin CDK sequence complex during the cell cycle from G1 to S in the cell line.

To confirm this, experiments were conducted using S284 point-related mutant cells (FOX-S284A, FOX-S248D), Mock (a vector transformed with a vector not containing the FOXO3 gene), and FOX-OV of the previously prepared FOXO3 did.

After dividing each cell with the same amount (10,000 cells), the total number of cells was measured every 24 hours.

As shown in FIGS. 8A and 8B, it was confirmed that the number of cells was changed according to the change of saccharification at S284 of FOXO3 from 5 days after transfection in which cell number change was observed optimally. When no saccharification occurred in the S284 region (FOX-S284A) or phosphorylation proceeded (FOX-S284D), the total number of cells did not increase. This means that the glycosylation at S284 of FOXO3 is essential for the proliferation of cancer cells (PDAC cells). The result of WST-1 analysis, FIG. 8B, also showed that FOX-S284A and FOX-S284D decreased the proliferation of cancer cells compared with FOX-OV cells. At the same time, the FOX-S284A and FOX-S284D cell populations showed increased numbers of FOX-OV cells in the case of FIG. 8C in which each cell was confirmed by crystal violet staining. Fixed results were shown. These results suggest that the binding of O- GlcNAc to FOXO3 S284 can turn the cell phenotype into highly proliferative by blocking FOXO3 tumor suppressor activity through p21 inhibition.

Since FOXO3 regulates cell cycle through p21 inhibition, the cell lines (Mock, FOX-OV, FOX-S284A and FOX-S284D) were treated with fluorescence activated cell sorter (FACS) The cell cycle was measured.

As a result, as shown in Figures 9a and 9b, the FOX-S284A cell line exhibited reduced proliferative activity through the maintenance of the active form of p21 which inhibits the expression of CDK2, cyclin D1, and cyclin E And induce G1 inhibition. FOX-S284D showed G2 arrest suggesting that O- GlcNAc glycation in FOXO3 S284 has an effect on the cell cycle pattern. That is, the above result shows that S284 is an important PTM site required to regulate the proliferation of pancreatic cancer cells, and O- GlcNAc glycosylation of S284 is more definite that it inhibits FOXO3 tumor suppressing activity through inhibition of p21 and induces cancer cell proliferation .

[ Example  5] After administration of pancreatic cancer treatment to pancreatic cancer cell line, Fork head box  O 3 (FOXO3) and O- GlcNAc glycation level analysis

To determine whether the anticancer agent is effective on the function of FOXO3 mutated with O- GlcNAc mutation (impaired tumor suppressive activity), the expression and glycosylation level of fork hair box O 3 (FOXO3) protein after administration of the therapeutic agent for pancreatic cancer to pancreatic cancer cell line After treatment with pancreatic cancer treatment, Western blot analysis was performed.

It was determined for 50% inhibition IC ₅₀ value or a density that is observed effect of each drug before it can process the cell line (Fig. 10a).

Fetal Bovine Serum (Gibco), a mixture of ⁶ HPX cells (Homo sapiens pancreas adenocarcinoma) with high FOXO3 O- GlcNAc glycosylation level and ⁶ Dulbecco's modified Eagle's medium (DMEM, Gibco) , 1% Penicillin-Streptomycin (Gibco)] in a 100 mm Petri dish. (Act D, Actinomycin D, Sigma), 10 nM paclitaxel (Pac, Paclitaxel, Sigma), 10 nM gemcitabine (Gem, Gemcitabine, Sigma), 10 nM doxorubicin (Dox, Doxorubicin, Sigma) Cells were treated with 10 nM salinomycin (Sal, Salinomycin, Sigma) and BG (OGT inhibitor), respectively, and cultured at 37 ° C and 5% CO ₂ for 24 hours. After 24 hours, the cells were transferred to a Dulbecco's modified Eagle medium mixture. Cells were cultured in the same manner except that Thiamet G was treated as a control and 50 μl of DMSO was treated as a negative control.

After immunoprecipitation analysis was performed on each cell line treated with the therapeutic agent for pancreatic cancer, the resultant was subjected to Western blotting using an anti-FOXO3 antibody. After 10% SDS-PAGE analysis, the entire gel was transferred to a nitrocellulose (NC) membrane for Western blotting, and then washed with TBS-T buffer solution containing 5% skimmed milk [20 mM Tris , 137 mM chloride-sodium, 0.1% Tween-20, pH 7.6] for 1 hour. Anti- O- GlcNAc (RL2, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) The pear was subjected to secondary treatment. The final NC membrane was reacted with ECL Plus Western blotting reagent (GE Healthcare) for 1 minute and analyzed by scanning with a Typhoon 9400 scanner.

As a result, as shown in Figs. 10B, 10C, and 10D, the O- glycNAc glycation level of the fork hair box O 3 (FOXO3) protein decreased in the pancreatic cancer cell line in which most of the pancreatic cancer therapeutic agents except for the control group (DMSO) Level. This means that the anticancer drug inhibits the glycosylation of FOXO3 of cancer cells and inhibits the proliferation of cancer cells. Interestingly, gemcitabine, commonly used for the treatment of pancreatic cancer, dose-dependently reduced the level of O- GlcNAc glycation. This result is damage to the FOXO3 by site-specific O-GlcNAc glycosylation in S284 that promotes pancreatic cancer cell growth can be partially restored by a treatment with an anticancer agent which induces p21 activation, FOXO3 site-specific glycosylation of the protein O -GlcNAc ≪ RTI ID = 0.0 > biochemical < / RTI > Therefore, not only can the sensitivity of the anticancer agent be confirmed using the same, but it can also be applied to the search for a substance having anticancer activity according to the degree of O- GlcNAc glycosylation of the FOXO3 protein.

<110> UNIVERSITY-INDUSTRY FOUNDATION, YONSEI UNIVERSITY <120> USE OF FORKHEAD BOX O 3 PROTEIN <130> P17U16C1053 <150> KR 10-2016-0156311 <151> 2016-11-23 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 673 <212> PRT <213> Unknown <220> <223> FORKHEAD BOX O 3 PROTEIN <400> 1 Met Ala Glu Ala Pro Ala Ser Pro Ala Pro Leu Ser Pro Leu Glu Val   1 5 10 15 Glu Leu Asp Pro Glu Phe Glu Pro Gln Ser Arg Pro Ser Ser Cys Thr              20 25 30 Trp Pro Leu Gln Arg Pro Glu Leu Gln Ala Ser Pro Ala Lys Pro Ser          35 40 45 Gly Glu Thr Ala Ala Asp Ser Met Ile Pro Glu Glu Glu Asp Asp Glu      50 55 60 Asp Gly Asp Gly Gly Gly Arg Ala Gly Ser Ala Met Ala Ile Gly  65 70 75 80 Gly Gly Gly Gly Ser Gly Thr Leu Gly Ser Gly Leu Leu Leu Glu Asp                  85 90 95 Ser Ala Arg Val Leu Ala Pro Gly Gly Gln Asp Pro Gly Ser Gly Pro             100 105 110 Ala Thr Ala Aly Gly Aly Leu Gly         115 120 125 Pro Gln Gln Pro Leu Pro Pro Pro Gln Pro Gly Ala Ala Gly Gly Ser     130 135 140 Gly Gln Pro Arg Lys Cys Ser Ser Arg Arg Asn Ala Trp Gly Asn Leu 145 150 155 160 Ser Tyr Ala Asp Leu Ile Thr Arg Ala Ile Glu Ser Ser Pro Asp Lys                 165 170 175 Arg Leu Thr Leu Ser Gln Ile Tyr Glu Trp Met Val Arg Cys Val Pro             180 185 190 Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala Gly Trp Lys Asn         195 200 205 Ser Ile Arg His His Leu Ser Leu His Ser Arg Phe Met Arg Val Gln     210 215 220 Asn Glu Gly Thr Gly Lys Ser Ser Trp Trp Ile Ile Asn Pro Asp Gly 225 230 235 240 Gly Lys Ser Gly Lys Ala Pro Arg Arg Arg Ala Val Ser Met Asp Asn                 245 250 255 Ser Asn Lys Tyr Thr Lys Ser Arg Gly Arg Ala Ala Lys Lys Lys Ala             260 265 270 Ala Leu Gln Thr Ala Pro Glu Ser Ala Asp Ser Ser Ser Gln Leu         275 280 285 Ser Lys Trp Pro Gly Ser Pro Thr Ser Arg Ser Ser Asp Glu Leu Asp     290 295 300 Ala Trp Thr Asp Phe Arg Ser Ser Thr Asn Ser Asn Ala Ser Thr Val 305 310 315 320 Ser Gly Arg Leu Ser Pro Ile Met Ala Ser Thr Glu Leu Asp Glu Val                 325 330 335 Gln Asp Asp Asp Ala Pro Leu Ser Pro Met Leu Tyr Ser Ser Ser Ala             340 345 350 Ser Leu Ser Pro Ser Val Ser Lys Pro Cys Thr Val Glu Leu Pro Arg         355 360 365 Leu Thr Asp Met Ala Gly Thr Met Asn Leu Asn Asp Gly Leu Thr Glu     370 375 380 Asn Leu Met Asp Asp Leu Leu Asp Asn Ile Thr Leu Pro Pro Ser Gln 385 390 395 400 Pro Ser Pro Thr Gly Gly Leu Met Gln Arg Ser Ser Ser Phe Pro Tyr                 405 410 415 Thr Lys Gly Ser Gly Leu Gly Ser Pro Thr Ser Ser Phe Asn Ser             420 425 430 Thr Val Phe Gly Pro Ser Ser Leu Asn Ser Leu Arg Gln Ser Pro Met         435 440 445 Gln Thr Ile Gln Glu Asn Lys Pro Ala Thr Phe Ser Ser Ser Met His     450 455 460 Tyr Gly Asn Gln Thr Leu Gln Asp Leu Leu Thr Ser Asp Ser Leu Ser 465 470 475 480 His Ser Asp Val Met Met Thr Gln Ser Asp Pro Leu Met Ser Gln Ala                 485 490 495 Ser Thr Ala Val Ser Ala Gln Asn Ser Arg Arg Asn Val Met Leu Arg             500 505 510 Asn Asp Pro Met Met Ser Phe Ala Ala Gln Pro Asn Gln Gly Ser Leu         515 520 525 Val Asn Gln Asn Leu Leu His His Gln His Gln Thr Gln Gly Ala Leu     530 535 540 Gly Gly Ser Arg Ala Leu Ser Asn Ser Val Ser Asn Met Gly Leu Ser 545 550 555 560 Glu Ser Ser Ser Leu Gly Ser Ala Lys His Gln Gln Gln Ser Ser Val                 565 570 575 Ser Gln Ser Met Gln Thr Leu Ser Asp Ser Leu Ser Gly Ser Ser Leu             580 585 590 Tyr Ser Thr Ser Ala Asn Leu Pro Val Met Gly His Glu Lys Phe Pro         595 600 605 Ser Asp Leu Asp Leu Asp Met Phe Asn Gly Ser Leu Glu Cys Asp Met     610 615 620 Glu Ser Ile Ile Arg Ser Glu Leu Met Asp Ala Asp Gly Leu Asp Phe 625 630 635 640 Asn Phe Asp Ser Leu Ile Ser Thr Gln Asn Val Val Gly Leu Asn Val                 645 650 655 Gly Asn Phe Thr Gly Ala Lys Gln Ala Ser Ser Gln Ser Trp Val Pro             660 665 670 Gly    

Claims (19)

GlcNAc glycosylation of an amino acid residue selected from the group consisting of S284, S286, T475, S482, S551 and combinations thereof in the amino acid sequence of the forkhead box O3 (FOXO3) protein of SEQ ID NO: 1 How to analyze the level of FOXO3 protein. The method according to claim 1,
Wherein the O-GlcNAc glycosylation of the protein further probes the O-GlcNAc glycosylation of an amino acid residue selected from the group consisting of S411, S476 and combinations thereof in the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein said O- GlcNAc glycosylation is performed using an antibody that specifically recognizes O- GlcNAc glycation. Contacting a candidate material with a sample comprising cells expressing a forkhead box O 3 (FOXO3) protein;
Measuring the O- GlcNAc glycosylation level of the forkhead box O 3 (FOXO3) protein of the sample; And
When the O-GlcNAc glycosylation level of the FOXO3 protein of the sample after contact with the candidate substance is lower than the O-GlcNAc glycation level of the FOXO3 protein of the sample before contact with the candidate substance, the candidate substance is judged to have anticancer activity &Lt; / RTI &gt;
Wherein the O-GlcNAc glycosylation of the FOXO3 protein is O-GlcNAc glycosylation of amino acid residues selected from the group consisting of S284, S286, T475, S482, S551 and combinations thereof in the amino acid sequence of SEQ ID NO: 1. 5. The method of claim 4,
Wherein the cell expressing the forkhead box O 3 (FOXO3) protein is selected from the group consisting of a pancreatic cancer cell line, a gastric cancer cell line, a liver cancer cell line and a combination thereof. 5. The method of claim 4,
Further comprising determining whether the amino acid residues selected from the group consisting of S411, S476, and combinations thereof in the amino acid sequence of the FOXO3 protein of SEQ ID NO: 1 are O-GlcNAc glycosylated. An antibody specifically recognizing the O- glycNAc glycosylation of amino acid residues selected from the group consisting of S284, S286, T475, S482, S551 residues and combinations thereof in the amino acid sequence of the forkhead box O3 (FOXO3) protein of SEQ ID NO: Or a pharmaceutically acceptable salt thereof. 8. The method of claim 7,
Wherein said composition further comprises an antibody that specifically recognizes the O-GlcNAc glycosylation of amino acid residues selected from the group consisting of S411, S476, and combinations thereof in the amino acid sequence of SEQ ID NO: 1. Measuring the O-GlcNAc glycosylation level of the forkhead box O3 (FOXO3) protein in a sample separated from the subject,
Wherein the O-GlcNAc glycosylation of the FOXO3 protein comprises determining the O-GlcNAc glycosylation of an amino acid residue selected from the group consisting of S284, S286, T475, S482, S551, and combinations thereof in the amino acid sequence of SEQ ID NO: To predict the prognosis. 10. The method of claim 9,
The O- GlcNAc glycation level of FOXO3 protein and the O- GlcNAc glycation level of FOXO3 protein in samples isolated from the individual after chemotherapy were compared in the samples separated from the individual before chemotherapy, and the O- GlcNAc And determining that an anticancer treatment is effective when the level of glycation is lower. Treating the sample separated from the subject with an anticancer agent;
And measuring the O- glycNAc glycosylation level of the fork hair box O 3 (FOXO3) protein of the anticancer agent-treated sample,
To determine the O-GlcNAc glycosylation level of the FOXO3 protein, it is necessary to determine the O-GlcNAc glycosylation of amino acid residues selected from the group consisting of S284, S286, T475, S482, S551 and combinations thereof in the amino acid sequence of SEQ ID NO: 1 A method for confirming susceptibility of an individual to an anticancer agent. 12. The method of claim 11,
When the O- glycNAc glycation level of the FOXO3 protein and the O- GlcNAc glycation level of the FOXO3 protein after the anticancer treatment were compared with each other before the anticancer treatment, the O- glycNAc glycation level of the FOXO3 protein after the anticancer treatment was lower, Classifying it as having; A method for confirming susceptibility to an anticancer agent of an object further comprising A composition for anti-cancer comprising a substance which inhibits the O- glycNAc glycosylation of S284 residue in the amino acid sequence of the fork hair box O 3 (FOXO3) protein of SEQ ID NO: 1. 14. The method of claim 13,
Wherein said substance further inhibits O-GlcNAc glycosylation of an amino acid residue selected from the group consisting of S286, S411, T475, S476, S482, S551 and combinations thereof in the amino acid sequence of SEQ ID NO: 1. The method according to claim 13 or 14,
Wherein the substance is an antibody specific for the S284 residue or the O-GlcNAc glycosylated S284 residue in the amino acid sequence of the forkhead box O3 (FOXO3) protein of SEQ ID NO: 1. Measuring the O-GlcNAc glycosylation of the fork head box O 3 (FOXO3) protein in a sample separated from the subject,
The O-GlcNAc glycosylation assay of the FOXO3 protein comprises determining the O-GlcNAc glycosylation of amino acid residues selected from the group consisting of S284, S286, T475, S482, S551, and combinations thereof in the amino acid sequence of SEQ ID NO: The method comprising the steps of: An antibody specifically recognizing the O-GlcNAc glycosylation of an amino acid residue selected from the group consisting of S284, S286, T475, S482, S551 and combinations thereof in the amino acid sequence of the forkhead box O 3 (FOXO3) protein of SEQ ID NO: &Lt; / RTI &gt; 18. The method of claim 17,
Wherein said antibody further comprises an antibody that specifically recognizes the O-GlcNAc glycosylation of amino acid residues selected from the group consisting of S411, S476, and combinations thereof in the amino acid sequence of SEQ ID NO: 1. An antibody that specifically recognizes a fox headbox O 3 (FOXO3) protein; And an antibody that specifically recognizes the O-GlcNAc glycosylation of the FOXO3 protein,
An antibody that specifically recognizes the O-GlcNAc glycosylation of the FOXO3 protein comprises an O-GlcNAc of amino acid residues selected from the group consisting of S284, S286, T475, S482, S551, and combinations thereof in the amino acid sequence of the FOXO3 protein of SEQ ID NO: Which specifically recognizes glycation.

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