Biomarker composition comprising hornerin for predicting prognosis of cancer, and use thereof

Abstract

The present invention provides a novel biomarker capable of predicting the prognosis of cancer patients or determining the suitability of anticancer therapy in order to select an appropriate anticancer therapy for cancer patients. By considering that death in mitosis (DiM) occurs during mitosis in most cancer cells if a DNA damage response, which is one of the mechanisms of anticancer therapy, is induced in cancer cells, it is shown that hornerin is related to DiM during mitosis, having occurred because of a DNA damage response, and it is identified that the expression level of hornerin is related to the prognosis of cancer patients, and thus hornerin is provided as a biomarker for predicting the prognosis of cancer patients or assessing the suitability of anticancer therapy.

Description

Biomarker composition for predicting the prognosis of cancer including hornerin and use thereof

[0001] The present invention relates to a biomarker composition for predicting the prognosis of cancer, including hornerin, and its use.

[0002] The DNA damage response (DDR) transmits signals to cell cycle checkpoints to either repair DNA or initiate cell death, preventing the transmission of damaged DNA to offspring. During mitosis, DNA repair enzymes recognize mitotic telomeres as genetic damage and fuse them, inhibiting key DNA repair processes. Despite the checkpoint-mediated DNA protection during the cell cycle, excessive DNA damage or a weakened G2 / M checkpoint can lead to premature initiation of mitosis or inappropriate mitosis, resulting in death in mitosis (DiM) or mitotic catastrophe. DDR is known to induce cell death in the G1 phase through the ATR-Chk1-Aurora B pathway, but the specific mechanism by which it induces death in mitosis (DiM) remains unknown.

[0003] The centrosome consists of two centrioles arranged at right angles and the pericentriolar material (PCM), which forms microtubules. The microtubules formed from the centrosome are used for cell movement in interphase, for forming cell polarity to prepare for mitosis, or for separating sister chromatids during mitosis. The replication of the centrioles that make up the centrosome is replicated once in the S phase of the cell cycle, similar to DNA replication. The replication of centrioles is initiated by the assembly of Plk4, STIL, and SAS-6 at the wall of the mother centriole. The replicated centriole pair separates in G2 to form a bipolar spindle, and in anaphase when chromosome separation is complete, they are separated by the cohesin-degrading enzyme separase. Here, Sgo1 and Plk1 protect the premature separation of centrioles in a manner similar to the protective process of cohesin removal by Sgo1 and several kinases. Therefore, overexpression of the kinases or depletion of Sgo1 leads to unscheduled chromosome segregation or premature centriole disengagement. When centriole duplication and separation do not proceed normally, phenomena such as centriole overduplication or centriole disengagement occur, and when these events occur, the cell forms a multipolar spindle.

[0004] Several DNA-damaging agents (DDA) used as anticancer agents have been reported to induce multipolar spindles in cancer cells, but the mechanism by which multipolar spindles are formed during the cell cycle is still unclear.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] Korean Patent Publication No. 10-2018-0054657 (Published on May 24, 2018)

[0008] The purpose of the present invention is to provide a new biomarker that can predict the prognosis of cancer patients or determine the suitability of anticancer treatment in order to select appropriate anticancer treatment for cancer patients. Referring to the fact that most cancer cells undergo death in mitosis (DiM) when the DNA damage response, which is one of the mechanisms of anticancer treatment, is induced in cancer cells, the present invention proves that hornerin is related to death in mitosis (DiM) that progresses through the DNA damage response, and confirms that the expression level of hornerin is related to the prognosis of cancer patients, thereby providing hornerin as a biomarker for predicting the prognosis of cancer patients or for screening the suitability of anticancer treatment.

[0009] The present invention provides a biomarker composition for predicting the prognosis of a cancer patient, including hornerin.

[0010] In addition, the present invention provides a biomarker composition for screening suitability for anticancer treatment including Hornerin.

[0011] In addition, the present invention provides a composition for predicting the prognosis of a cancer patient, comprising a preparation for measuring the expression level of Hornerin.

[0012] Additionally, the present invention provides a kit for screening suitability for anticancer treatment, comprising a preparation for measuring the expression level of Hornerin.

[0013] In addition, the present invention provides a method for providing information for predicting the prognosis of a cancer patient, comprising the steps of: measuring the mRNA expression level or protein expression level of Hornerin in a biological sample isolated from a cancer patient; and predicting that the patient's prognosis will be good if the mRNA expression level or protein expression level of Hornerin is higher than that of a control sample.

[0014] Additionally, the present invention provides a method for providing information for determining the suitability of anticancer treatment, comprising the step of measuring the level of Hornerin in a biological sample isolated from a cancer patient prior to receiving anticancer treatment.

[0015] According to the present invention, even if a DNA damage response is induced in cancer cells, if hornerin is absent, multipolar spindles cannot be formed, death in mitosis (DiM) does not proceed, and cells undergo cell division with damaged DNA. Therefore, it can be determined that anticancer drug treatment or radiation therapy utilizing the DNA damage response is suitable only when hornerin is sufficiently present. In addition, according to the present invention, it has been confirmed that patients with cervical cancer, pancreatic cancer, or renal cancer who have a low expression level of hornerin have a worse prognosis than patients with a high expression level. Therefore, the prognosis of cancer patients can be predicted based on the expression level of hornerin.

[0016] Figure 1 is a schematic diagram showing the role of Hornerin in death in mitosis (DiM) that progresses as a DNA damage response according to the present invention.

[0017] Figure 2 is a live cell imaging result confirming the change in cell cycle caused by DNA damage response induced in cancer cells.

[0018] Figure 3 shows the results of analyzing the proportion of bipolar spindles or multipolar spindles in cancer cells that underwent death in mitosis (DiM) among cancer cells in which a DNA damage response was induced.

[0019] Figure 4 shows the results of analyzing the proportion of cells that undergo death in mitosis (DiM) or undergo regression of cytoplasmic inclusions and progress to binucleate cells among cancer cells that exhibit multipolar spindles in which a DNA damage response is induced.

[0020] Figure 5 shows the results of analyzing the effect of DNA damage response on the shape of the spindle in cells that have progressed to prometaphase.

[0021] Figure 6 shows the results of analyzing the proportion of cells exhibiting multipolar spindles in cells in which the DNA damage response progressed to prometaphase.

[0022] Figure 7 shows the results of analyzing the effects of inhibitors of factors related to the DNA damage response on multipolar spindles induced by the DNA damage response.

[0023] Figure 8 shows the results of analyzing the effect of inhibitors of factors related to the DNA damage response on the change in the proportion of prometaphase cells exhibiting multipolar spindles induced by the DNA damage response.

[0024] Figure 9 shows the results of an immunofluorescence image analysis of the effect of the DNA damage response on the number of centrosomes.

[0025] Figure 10 shows the results of analyzing the effect of DNA damage response on the number of centrioles in mitotic cells.

[0026] Figure 11 shows the results of immunoblotting analysis of the effects of inhibitors of factors related to the DNA damage response on the expression levels of key factors in the initiation of centriole replication in the DNA damage response.

[0027] Figure 12 shows the results of immunostaining analysis of the effects of inhibitors of factors related to the DNA damage response on the expression levels of key factors in the initiation of centriole replication in the DNA damage response.

[0028] Figure 13 shows the results of live cell imaging analysis to determine whether the DNA damage response results in premature centriole disengagement.

[0029] Figure 14 shows the results of analyzing the effect of the DNA damage response on the positioning of centrin 1 and ninein in relation to premature centriole disengagement.

[0030] Figure 15 shows the results of analyzing the effect of the DNA damage response on the position of centrin 1 and β-tubulin in relation to premature centriole disengagement.

[0031] Figure 16 shows the results of analyzing the proportion of cells with disengaged centrioles in cells that formed multipolar spindles in response to DNA damage.

[0032] Figure 17 shows the results of analyzing the effect of inhibitors of factors related to the DNA damage response on the proportion of cells with centriole disengagement induced by the DNA damage response.

[0033] Figure 18 shows the results of immunofluorescence analysis of the effect of DNA damage response on centriole overduplication.

[0034] Figure 19 shows the results of analyzing the effect of DNA damage response on the proportion of cells with centriole overduplication.

[0035] Figure 20 shows the results of suppressing the expression of endogenous separase using siRNA and confirming the expression of recombinant separase using a Myc-separase expression plasmid.

[0036] Figure 21 shows the results of analyzing the effect of separase expression on the proportion of cells with centriole disengagement induced by the DNA damage response.

[0037] Figure 22 shows the results of analyzing the effect of DNA damage response on the expression level of Plk1.

[0038] Figure 23 shows the results of analyzing the effect of DNA damage response on the expression level of Sgo1.

[0039] Figure 24 shows the results of Western blotting analysis of the effect of DNA damage response on the movement of Plk1 and Sgo1 to the centrosome.

[0040] Figure 25 shows the results of IP-immunoblotting analysis of the effect of DNA damage response on the interaction between Plk1 and Sgo1.

[0041] Figure 26 shows the results of a pull-down assay analyzing the effect of the DNA damage response on the interaction between Plk1 and Sgo1.

[0042] Figure 27 shows the results of analyzing the effect of phosphorylation of the polo-box domain (PBD) of Plk1 on the formation of multipolar spindles induced by the DNA damage response.

[0043] Figure 28 shows the results of analyzing the effect of polo-box domain (PBD) phosphorylation of Plk1 on centrosome localization following DNA damage response.

[0044] Figure 29 shows the results of analyzing the effect of phosphorylation of the polo-box domain (PBD) of Plk1 on the cellular proportion of multipolar spindles according to the DNA damage response.

[0045] Figure 30 shows the results of identifying Plk1 phosphorylation sites affected by the DNA damage response and analyzing the cell cycle by the amounts of Plk1, Chk1, cyclin A, and cyclin B1.

[0046] Figure 31 shows the results of analyzing Plk1 phosphorylation in the G2 phase induced by a Cdk1 inhibitor, and confirming entry into the G2 phase and M phase at the levels of cyclin A and cyclin B1.

[0047] Figure 32 shows the results of analyzing the effects of inhibitors of factors related to the DNA damage response on Plk1 phosphorylation induced by the DNA damage response.

[0048] Figure 33 shows the results of confirming the site of Plk1 phosphorylated by Cdk1.

[0049] Figure 34 shows the results of analyzing the effect of autophosphorylation of active Plk1 on Cdk1-induced Plk1 phosphorylation.

[0050] Figure 35 shows the results of confirming whether Cdk1 directly interacts with Plk1.

[0051] Figure 36 shows that the inhibitor of cell division-related factors is Ser of Plk1. 526 This is the result of analyzing the effect on phosphorylation.

[0052] Figure 37 shows Aurora A in Plk1 and Ser 526 This is the result of analyzing whether phosphorylation occurs.

[0053] Figure 38 shows the results of analyzing whether the phosphorylation of Plk1 affects its interaction with Sgo1.

[0054] Figure 39 shows the results of IP-immunoblotting analysis of the effect of hornerin on the interaction between Chk1 and Plk1 due to DNA damage response.

[0055] Figure 40 shows the results of analyzing the effect of the presence or absence of hornerin on the reduced Plk1 level in the centrosome due to the DNA damage response.

[0056] Figure 41 shows the results of analyzing the effect of the presence or absence of hornerin on the reduced Sgo1 level in the centrosome due to the DNA damage response.

[0057] Figure 42 shows the effect of hornerin on Thr of Plk1 induced by DNA damage response. 210 , Ser 526 , Ser 529 or Thr 539 This is the result of analyzing the effect on phosphorylation.

[0058] Figure 43 shows the results of analyzing the effect of the presence or absence of hornerin on the formation of the Cep192::Aurora A::Plk1 complex.

[0059] Figure 44 is Ser of Plk1 526 , Ser 529 and Thr 539 The results of an in vitro analysis of the interaction with hornerin depending on the presence or absence of phosphorylation.

[0060] Figure 45 is Ser of Plk1 526 , Ser 529 and Thr 539 The results of in vitro analysis of the interaction with Cep192 depending on phosphorylation status.

[0061] Figure 46 is Ser of Plk1 526, Ser 529 and Thr 539 The results of in situ analysis of the interaction with hornerin depending on phosphorylation status.

[0062] Figure 47 shows the results of analyzing the intracellular location of hornerin according to the cell cycle.

[0063] Figure 48 shows Ser upon depletion of hornerin. 526 , Ser 529 and Thr 539 This is the result of analyzing whether phosphorylated mutant Plk1 moves to the centrosome.

[0064] Figure 49 shows the results of analyzing the effect of DNA damage response on the level of Aurora A in the centrosome.

[0065] Figure 50 shows the results of analyzing the effect of DNA damage response on the level of Cep192 in the centrosome.

[0066] Figure 51 is Ser of Plk1 526 , Ser 529 and Thr 539 This is the result of analyzing the change in the level of Cep192 in the centrosome depending on the presence or absence of phosphorylation.

[0067] Figure 52 is Ser of Plk1 526 , Ser 529 and Thr 539 This is the result of analyzing the change in the level of Sgo1 in the centrosome depending on the presence or absence of phosphorylation.

[0068] Figure 53 shows the results of analyzing changes in the level of Plk1 in the centrosome according to phosphorylation mutations of Plk1.

[0069] Figure 54 shows the results of analyzing the changes in the specific gravity of prometaphase cells exhibiting multipolar spindles according to phosphorylation mutations of Plk1.

[0070] Figure 55 is Ser of Plk1526 , Ser 529 and Thr 539 Phosphorylation of Thr, an activator of Plk1 210 This is the result of analyzing the effect on phosphorylation.

[0071] Figure 56 shows the results of analyzing the effect of the presence or absence of Hornerin on changes in the size and number of cell colonies induced by the DNA damage response.

[0072] Figure 57 shows the results of analyzing the effect of the presence or absence of Hornerin on the change in the size of cell colonies induced by the DNA damage response.

[0073] Figure 58 shows the results of analyzing the effect of the presence or absence of Hornerin on the change in the number of cell colonies induced by the DNA damage response.

[0074] Figure 59 shows the presence or absence of Hornerin in the PBD region of Plk1, which induces a DNA damage response. 526 , Ser 529 and Thr 539 This is the result of analyzing the effect on phosphorylation.

[0075] Figure 60 shows the results of analyzing the change in survival rate of patients with cervical cancer, pancreatic cancer, or renal cancer according to the expression level of Hornerin in the TCGA database.

[0076] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0077] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0078] Hereinafter, the present invention will be described in more detail.

[0079] According to the present invention, when the DNA damage response, which is one of the mechanisms of anticancer treatment, is induced in cancer cells, death in mitosis (DiM) progresses in most cancer cells. Therefore, it has been proven that hornerin mediates death in mitosis (DiM) progressing through the DNA damage response. Therefore, by examining the expression level of hornerin, it is possible to determine whether a cancer patient is suitable for anticancer treatment utilizing the DNA damage response.

[0080] Specifically, when a DNA damage response is induced by anticancer drugs or radiation therapy, the proportion of cells in which multipolar spindles are observed increases, and it was confirmed that the formation of multipolar spindles induced by the DNA damage response is due to premature centriole disengagement. The premature centriole disengagement is associated with a decrease in the levels of Plk1 and Sgo1, and the decrease in Plk1 expression is mediated by Chk1, and the DNA damage response induces premature centriole disengagement by interfering with the interaction between Plk1 and Sgo1. The Chk1-mediated decrease in Plk1 and the inhibition of the interaction between Plk1 and Sgo1 are caused by Ser 526 , Ser 529 and Thr 539 It was confirmed that it was related to phosphorylation, and hornerin was involved in the phosphorylation process of Plk1 by Chk1 (Fig. 1).

[0081] Ultimately, in cells where the DNA damage response is induced, hornerin mediates Ser in the PBD region of Plk1. 529 and Thr 539 Phosphorylation of Ser is induced, and when these cells enter mitosis, Ser is induced by Aurora A. 529When this phosphorylation occurs, Plk1 with phosphorylated PBD site maintains a conformational change and an inactive state, thereby preventing the translocation of Cep192::Aurora A::Plk1 complex and Sgo1 to the centrosome, thereby inducing death in mitosis (DiM) involving multipolar spindles. Furthermore, by demonstrating that the presence or absence of hornerin affects death in mitosis (DiM) induced by the DNA damage response, we demonstrated that the presence of hornerin is essential for the anticancer response through the DNA damage response. In addition, by confirming that changes in the expression level of hornerin are related to the survival rate of various cancer patients, we demonstrated that the expression level of hornerin can be a biomarker that can predict the prognosis of cancer patients.

[0082] The term "biomarker" used in the present invention refers to a substance that can diagnose a biological sample isolated from a subject suffering from a disease by distinguishing it from a biological sample of a normal control group, and includes organic biomolecules such as proteins, nucleic acids, lipids, glycolipids, and glycoproteins that show an increase or decrease in the biological sample of a subject suffering from a disease compared to the normal control group. The biological sample refers to tissue, blood, plasma, urine, or cells isolated from the subject.

[0083] The term "prognosis prediction" as used herein refers to the act of predicting the course and outcome of a disease in advance. More specifically, prognosis prediction can be interpreted as any act of predicting the course of a disease after treatment by comprehensively considering the patient's physiological and environmental conditions, as the course of the disease after treatment can vary depending on the patient's physiological and environmental conditions.

[0084] For the purposes of the present invention, the above prognosis prediction can be interpreted as an act of predicting the disease-free survival rate or survival rate of a cancer patient by predicting the course of the disease and whether or not the cancer patient will be cured after drug treatment. For example, predicting a "good prognosis" means that the disease-free survival rate or survival rate of the cancer patient after drug treatment is high, indicating a high possibility of the cancer patient being cured, and predicting a "poor prognosis" means that the disease-free survival rate or survival rate of the cancer patient after drug treatment is low, indicating a high possibility of the cancer patient recurring or dying from the cancer.

[0085] The present invention provides a biomarker composition for predicting the prognosis of a cancer patient, including hornerin.

[0086] The above hornerin is a protein consisting of 2850 amino acids represented by sequence number 1, and is known to have the typical structural characteristics of the "fusion" S100 protein family isolated from skin. Hornerin is not detected in normal epithelial cells, but it has been reported that hornerin is detected during the skin regeneration process after injury.

[0087] In the present invention, the cancer patient is a patient who has received or will receive anticancer treatment using a DNA damage response, and the anticancer treatment is anticancer drug treatment or radiation therapy.

[0088] The above anticancer drugs are chemical substances that induce DNA damage response in cancer cells, specifically cisplatin, doxorubicin, etoposide, fluoropyrimidine, oxaliplatin, campthotecan, belotecan, cyclophosphamide, methotrexate, camptothecin, 5-fluorouracil (5-FU), floxuridine, gemcitabine, streptozocin, carboplatin, topotecan, irinotecan, hydroxyurea, meclorethamine, Chlorambucil, Busulfan, Doxifluridine, Mitomycin, Mitoxantron, Cyclophosphamide, or Mechlorethamine may be used, but are not limited thereto. The above-mentioned radiation therapy is a treatment method that induces a DNA damage response within cancer cells by irradiating the cancerous area or the entire body with radiation at a level of 1 Gy to 5 Gy.

[0089] The biomarker composition may further include Plk1 in which the 526th serine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 is phosphorylated; Plk1 in which the 529th serine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 is phosphorylated; Plk1 in which the 539th threonine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 is phosphorylated; Plk1 in which the 529th serine and the 539th threonine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 are phosphorylated; or Plk1 in which the 526th serine, the 529th serine, and the 539th threonine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 are phosphorylated.

[0090] The above Plk1 (polo-like kinase 1) is a serine / threonine protein kinase consisting of 603 amino acids represented by sequence number 2 and has a size of 66 kDa. The above Plk1 has a kinase domain at the N terminus and a PBD (polo-box domain) region at the C terminus.

[0091] In the present invention, the PBD region Ser of the Plk1 526 Phosphorylation means Plk1 in which the 526th serine from the N-terminus in the amino acid sequence represented by sequence number 2 is phosphorylated, and the PBD site Ser of Plk1 529 Phosphorylation means Plk1 in which the 529th serine from the N-terminus in the amino acid sequence represented by sequence number 2 is phosphorylated, and the PBD site Thr of the Plk1 539Phosphorylation means Plk1 in which the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2 is phosphorylated, and the PBD site Ser of the Plk1 529 and Thr 539 Phosphorylation means Plk1 in which the 529th serine and the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2 are phosphorylated, and the PBD site Ser of Plk1 526 , Ser 529 and Thr 539 Phosphorylation refers to Plk1 in which the 526th serine, the 529th serine, and the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2 are phosphorylated.

[0092] In addition, the present invention provides a biomarker composition for screening suitability for anticancer treatment including Hornerin.

[0093] In the present invention, in cells where a DNA damage response is induced, hornerin is mediated to Ser in the PBD region of Plk1. 526 , Ser 529 and Thr 539 By confirming that death in mitosis (DiM) progresses when phosphorylation of Plk1 is induced, Ser in the PBD region of Plk1 is induced in cancer cells. 526 , Ser 529 and Thr 539 Since phosphorylation of hornerin indicates whether death in mitosis (DiM) occurs due to DNA damage response, the presence or absence of hornerin and Ser in the PBD region of Plk1 are important. 526 , Ser 529 and Thr 539It was demonstrated that it is possible to determine whether anticancer treatment using the DNA damage response is possible depending on whether or not the phosphorylation of DNA is present.

[0094] The present invention also provides a composition for predicting the prognosis of a cancer patient, comprising a preparation for measuring the expression level of hornerin. The present invention also provides a kit for screening for suitability for anticancer treatment, comprising a preparation for measuring the expression level of hornerin.

[0095] The agent for measuring the above expression level may be a primer or probe that specifically binds to a gene encoding Hornerin, or an antibody, peptide, aptamer or compound that specifically binds to Hornerin.

[0096] The composition for predicting the prognosis of the cancer patient or the kit for screening the suitability of anticancer treatment may further include an agent for measuring phosphorylation of Plk1 that measures the phosphorylation level of the 526th serine, the 529th serine or the 539th threonine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2, and the agent for measuring phosphorylation of Plk1 may be an antibody, a peptide, an aptamer or a compound that specifically binds to the phosphorylation of the 526th serine, the 529th serine or the 539th threonine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2. In addition, since the mitotic cell death caused by the DNA damage response mediated by hornerin is due to a decrease in the amount of Plk1 in the centrosome and this phenomenon is overcome by the overexpression of Plk1, the agent for measuring the amount of Plk1 may further be included.

[0097] The term "primer" used in the present invention refers to a short genetic sequence that serves as the starting point for DNA synthesis, and is an oligonucleotide synthesized for the purpose of diagnosis, DNA sequencing, etc. The primers are typically synthesized and used in lengths of 15 to 30 base pairs, but this may vary depending on the intended use, and may be modified by methylation, capping, etc. using known methods.

[0098] The term "probe" used in the present invention refers to a nucleic acid capable of specifically binding to mRNA, ranging from a few bases to several hundred bases in length, produced through enzymatic, chemical, or synthetic separation and purification processes. The presence or absence of mRNA can be confirmed by labeling with a radioactive isotope or enzyme, and the probe can be designed and modified using known methods.

[0099] The term "antibody" used in the present invention is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the marker protein of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes special antibodies such as humanized antibodies.

[0100] The term "peptide" used in the present invention has the advantage of high binding affinity to target substances and is resistant to denaturation even during heat and chemical treatments. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0101] The term "aptamer" used in the present invention refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristic of being able to bind to a target molecule with high affinity and specificity. As described above, an aptamer can specifically bind to an antigenic substance in the same way as an antibody, but is composed of a polynucleotide that is more stable than a protein, has a simpler structure, and is easy to synthesize, and therefore can be used as a substitute for an antibody.

[0102] The term "kit" used in the present invention may include an antibody that specifically binds to a biomarker component, a secondary antibody conjugate to which a label that develops color by reaction with a substrate is conjugated, a chromogenic substrate solution that reacts with the label to develop color, a washing solution, an enzyme reaction stop solution, and the like, and may be manufactured into a plurality of separate packagings or compartments containing the reagent components used.

[0103] In addition, the present invention provides a method for providing information for predicting the prognosis of a cancer patient, comprising the steps of: measuring the mRNA expression level or protein expression level of Hornerin in a biological sample isolated from a cancer patient; and predicting that the patient's prognosis will be good if the mRNA expression level or protein expression level of Hornerin is higher than that of a control sample.

[0104] The method for providing information for predicting the prognosis of the cancer patient may further include the steps of: measuring whether phosphorylation occurs at the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 consisting of an amino acid sequence represented by SEQ ID NO: 2 in a biological sample isolated from the cancer patient; and, if phosphorylation occurs at the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 consisting of an amino acid sequence represented by SEQ ID NO: 2, predicting that the patient's prognosis will be good.

[0105] The above cancer patient is a patient who has received or will receive anticancer treatment using DNA damage response, and the above biological sample is tumor tissue.

[0106] Additionally, the present invention provides a method for providing information for determining the suitability of anticancer treatment, comprising the step of measuring the level of Hornerin in a biological sample isolated from a cancer patient prior to receiving anticancer treatment.

[0107] The above biological sample is tumor tissue, and if hornerin is detected in the biological sample, it can be determined that anticancer treatment is appropriate.

[0108] In addition, the method for providing information for determining the suitability of the anticancer treatment may further include a step of measuring whether phosphorylation of the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 consisting of an amino acid sequence represented by SEQ ID NO: 2 is present in a biological sample isolated from a cancer patient, and if phosphorylation of the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 is detected in the biological sample, it may be determined that the anticancer treatment is suitable.

[0109] Hereinafter, to aid understanding of the present invention, experimental examples and examples will be described in detail. However, the following experimental examples and examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental examples and examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0110]

[0111] <Experimental Example> Experimental Materials and Methods

[0112] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0113]

[0114] 1. Plasmid

[0115] The GFP-tagged Sgo1 expression vector, the Myc-tagged Separase expression vector, and the pCer-C3-Plk1-K82R vector were purchased from Addgene (Watertown, MA). Point mutations in Plk1 were generated using the Muta-direct™ Site Directed Mutagenesis kit (iNtRON BIOTECHNOLOGY, Seongnam, Korea) as follows: S526D, S529D, T539D, S526D / S529D, S526D / T539D, S529D / T539D, S526D / S529D / T539D (3D mutation), T210DS526D / S529D / S539D (4D mutation), T210A, S529A, T539A, or S526A / S529A / T539A (3A mutation). PCR fragments of the human Plk1 or Sgo1 gene were inserted into the PET-28a vector (Novagen, Darmstadt, Germany) to generate His-Plk1 or His-Sgo1 vectors.

[0116]

[0117] 2. Antibodies

[0118] Phosphorylated Ser in Plk1 529 and phosphorylated Thr 539 Rabbit antibodies against phosphorylated Ser were generated using C-AIILHLSNG(p)SVQIN and QINFFQDH(p)TKLILC peptides, respectively (AbClon, Seoul, Korea). 526 Rabbit antibodies against were generated using the L(p)SNGSVQI-C peptide (GenScript, New Jersey, USA).

[0119] For Western Blotting, the following antibodies were used: cyclin A (B-8, 1:1000, Santa Cruz Biotechnology Inc., sc-271682), cyclin B1 (H-20, 1:1000, Santa Cruz Biotechnology Inc., sc-594), p38 MAPK (N-20, 1:1000, Santa Cruz Biotechnology Inc., sc-728), Sgo1 (1:1000, Thermo Scientific Pierce Antibodies, PA5-30869), β-tubulin E7 monoclonal antibody (1:1000, Developmental Studies Hybridoma Bank USA, E7), separase (XJ11-1812, 1:500, Abcam, ab16170), PLK4 (6H5, 1:500, Millipore, MABC544), SAS-6 (91.390.21, 1:100, Santa Cruz Biotechnology Inc., sc-81431), STIL (1:500, Abcam, ab89314), Chk1 (2G1D5, 1:2500, Cell Signaling Technology, #2360), c-Myc (9E10, 1:1000, Santa Cruz Biotechnology Inc., sc-40), HSP70 (3A3, 1:10000, Santa Cruz Biotechnology Inc., sc-32239), hornerin (1:500, NOVUS Biologicals, NBP1-80807), AhR (1:1000, Proteintech, 67785-1), purified Plk1 pT539 (1:500, AbClon), and purified Plk1 pS529 (1:500, AbClon).

[0120] For immunostaining, the following antibodies were used: Aurora B T232 (1:100, Rockland Immunochemicals, Inc., 600-401-677), Sgo1 (F-8, 1:100, Santa Cruz Biotechnology Inc., sc-393993), PLK1 (E-2, 1:100, Santa Cruz Biotechnology Inc., sc-55504), PLK4 (6H5, 1:500, Millipore, MABC544), SAS-6 (91.390.21, 1:100, Santa Cruz Biotechnology Inc., sc-81431), STIL (1:500, Abcam, ab89314), C-NAP1 (1:100, Proteintech, 14498-1-AP), VPS4B (UT292, 1:50, Sigma-Aldrich, ABS1656), ALIX (3A9, 1:100, Thermo Scientific Pierce Antibodies, MA1-83977), lamin B1 (EPR8985(B), 1:100, Abcam, ab194106), pericentrin (1:100, Proteintech, 22271-1-AP), β-tubulin E7 monoclonal antibody (1:100, Developmental Studies Hybridoma Bank USA, E7), γ-tubulin (GTU-88, 1:100, Sigma-Aldrich, T6557), c-Myc (9E10, 1:1000, Santa Cruz Biotechnology Inc., sc-40), centrin (1:100, Proteintech, 12794-1-AP), purified PLK1 pT539 (1:400, AbClon), purified PLK1 pS529 (1:200, AbClon), phospho-H2AX (1:200, Cell Signaling), phospho-MPM2 (1:1000, Millipore, 05-368), Alexa Fluor. ® 488 goat anti-rabbit IgG (1:100, Thermo Scientific Pierce Antibodies, A11034), Alexa Fluor ® 594 goat anti-mouse IgG (1:100, Thermo Scientific Pierce Antibodies, A11032), and Alexa Fluor ® 594 goat anti-rabbit IgG (1:100, Thermo Scientific Pierce Antibodies, A11037).

[0121]

[0122] 3. Cell culture and metastasis

[0123] HeLa and RPE1 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Invitrogen), 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2. All cell lines were tested for mycoplasma contamination by PCR. Fingerprinting of all cell lines by 'AmpplFLSTR identifiler PCR Amplification kit' was tested at the Korean Cell Line Bank, and siRNA was transfected into HeLa cells using DharmaFect 1 (Dharmacon, Inc.). DNA testing was performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions.

[0124]

[0125] 4. Cell treatment

[0126] To induce DNA damage reaction (DDR) in cancer cells, Hela cells, a cervical cancer cell line, were treated with 0.5 μM benzo[a]pyrene (B[a]P) (Sigma) and 2.5 μM B[a]P-7,8-diol-9,10-epoxide (BPDE) (Sigma) for 48 h or with 0.5 μM etoposide (Sigma) for 24 h. To assess the inhibitory response, cells were treated with the ATR inhibitor NU6027 (2 μM) (Sigma), the ATM inhibitor KU55933 (20 nM) (Sigma), the Chk1 inhibitor UCN-01 (300 nM) (Sigma), the Chk2 inhibitor (1 μM), or the DNA-PK inhibitor (0.5 μM). For synchronization in G2, cells were treated with the Cdk1 inhibitor RO3306 (1 μM) for 24 h. For inhibition of mitotic kinases, cells were treated with the Cdk1 inhibitor RO3306 (1 μM) for 5 h, the Plk1 inhibitor BI2536 (1 μM) for 1 h, the Aurora A inhibitor VX680 (5 μM) for 1 h, and the Aurora B inhibitor Hesperadin (2 μM) (Selleckem) for 5 h. For mitotic arrest, 100 ng / ml nocodazole was treated for 16 hours.

[0127]

[0128] 5. siRNA

[0129] The control siRNA was 5`-CGTACGCGGAATACTTCGATT-3`. The following siRNA sequences were used: siHornerin-A: 5`-GCAACAUGGUUCUACAUCAUU-3`, siHornerin-B: 5`- CUGUCUUUGGUCAACAUGAUU-3`, siSeparase: 5`-CAAGGUUAGAUUUAAUCCUUU-3`, siPlk1: 5`-CCUUGAUGAAGAAGAUCAC-3`, siAhR: 5`-CGUUAGAUGUUCCUCUGUG-3`.

[0130]

[0131] 6. Immunoprecipitation

[0132] Antibodies were coupled to Affi-Prep protein beads (Bio-Rad Laboratory) at a concentration of 0.3 mg / ml. 1 × 10 were added to 1 ml NP-40 lysis buffer (50 mM HEPES, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM GTA, 1 mM MgCl2, 0.5 mM DTT, 10 μg / ml leupeptin, 10 μg / ml pepstatin, and 10 μg / ml chymostatin). 7 Cells were lysed. To preserve phosphorylation, the lysis buffer was supplemented with 1 mM NaVO4 and 0.5 μM microcystin LR (Alexis Biochemicals). The analytes were centrifuged and reacted with protein A beads conjugated with preimmune rabbit IgG for 1 h at 4°C, followed by overnight reaction with protein A beads conjugated with specific antibodies at 4°C. After completion of the reaction, the beads were washed with lysis buffer, heated in Laemmli sample buffer for 3 min, and analyzed by SDS-PAGE (polyacrylamide gel electrophoresis).

[0133]

[0134] 7. Immunofluorescence and live cell imaging

[0135] HeLa cells were fixed with methanol at -20°C for 30 min. Alternatively, soluble proteins were extracted from cells with BRB80-T buffer (80 mM PIPS, pH 6.8, 1 mM MgCl2, 5 mM EGTA, and 0.5% Triton X-100), and then fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were permeabilized with PBS-BT (1 x PBS, 3% BSA, 0.1% Triton X-100) for 30 min at room temperature. Afterwards, they were reacted with primary and secondary antibodies diluted in PBS-BT. Cell images were acquired using ZEN2 software (Carl Zeiss, Germany) with a Zeiss Axiovert 200 M microscope equipped with a 1.4 NA plan-Apo 100x oil immersion lens and an HRm CCD camera. Deconvoluted images were acquired and analyzed using AutoDeblur v9.1 and AutoVisualizer v9.1 (AutoQuant Imaging). The inserts are represented as a single focal plane within a boxed region. All images in the panel were acquired under a constant exposure time for all channels. To eliminate size differences in each displayed region, the average intensity was calculated from a selected region.

[0136] For time-lapse microscopy, HeLa cells stably expressing GFP-Histone H2B or GFP-centrin2 were cultured in L-15 medium (Invitrogen) containing 10% FBS (Invitrogen) and 2 mM L-glutamine (Invitrogen). Cells were placed in a heated growth chamber at 37°C and observed using a Zeiss Axiovert 200 M microscope equipped with a 20x objective lens. Images were acquired every 3 minutes for 5 h using AxioVision 4.8.2 (Carl Zeiss).

[0137]

[0138] 8. Purification of expressed proteins

[0139] His6-tagged Plk1 wild type (WT), S529A mutant, T539A mutant, 3A mutant, 3D mutant, or Sgo1 were inserted into the pET-28 vector, and each vector was induced to be expressed in E. coli BL21 DE3 (Novagen). The strains expressing each vector were lysed with lysis buffer (50 mM Na2HPO4, 100 mM NaCl, 10% glycerol, and 10 mM imidazole), and the target proteins were isolated by adding Ni2+-NTA-agarose (Qiagen). The isolated target proteins were washed with washing buffer (50 mM Na2HPO4, 100 mM NaCl, 10% glycerol, and 20 mM imidazole), and each protein was purified from Ni2+-NTA-agarose (Qiagen) according to standard procedures.

[0140]

[0141] 9. Pull-down assay

[0142] To perform the in vitro pull-down assay, 1 μg of each protein (His-Plk1 WT, 3A, 3D, or His-Sgo1) was reacted with 20 μl of Ni2+-NTA-agarose beads (Qiagen) at 4°C for 1 h. After washing three times with wash buffer (50 mM Na2HPO4, 100 mM NaCl, 10% glycerol, 20 mM imidazole), the protein-bead complexes were incubated with the corresponding cell lysate (500 μg total protein) at 4°C for 1 h. After washing three times with wash buffer, Laemmli sample buffer was added and heated at 95°C for 5 min to denature the beads. The samples were analyzed by immunoblotting.

[0143]

[0144] 10. In vitro kinase assay

[0145] For Chk1 kinase assay, 1 μg of His-Plk1 WT, T210A, S526A, S529A, or T539A mutants were treated with 200 ng of human recombinant Chk1 kinase (SignalChem) and Aurora A (purified from asynchronous sf9 cells) and incubated in 50 μl of kinase buffer (25 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM MgCl2, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) containing 10 μM ATP at 37°C for 30 min. Phosphate incorporation into His-Plk1 was analyzed by SDS-PAGE and immunoblotting using specific phospho-specific antibodies.

[0146]

[0147] 11. Tandem purification and proteomics

[0148] HeLa S3 cells expressing GFP-S-Plk1 were prepared. The cells were lysed in 1 ml of NP-40 lysis buffer (50 mM Hepes, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, 1 mM NaVO4, 0.5 μM microcystin LR, 5 mM C4H7NaO2, 10 μg / ml leupeptin, 10 μg / ml pepstatin, and 10 μg / ml chymostatin). The lysate was centrifuged and incubated overnight at 4°C with protein A beads conjugated with anti-GFP antibody. Antibody beads were recovered by centrifugation, washed five times with lysis buffer containing 500 mM KCl, washed twice with W / O protease inhibitor lysis buffer, and incubated overnight with TEV protease. S-Plk1 was isolated from protein A beads and GFP antibody by treatment. Protease inhibitors were added to the supernatant, and the beads were reacted with S-protein agarose beads (Sigma) at 4°C for 3 h. After the reaction was completed, the beads were washed five times with lysis buffer containing 0.5 mM DTT and treated with urea elution buffer (50 mM Tris pH 8.5, 8 M urea) for 30 min at room temperature. The purified protein complex was analyzed by a quantitative analysis method.

[0149]

[0150] 12. In situ proximity ligation assay (PLA)

[0151] Plasmid-transfected cells were treated with Duolink blocking solution (Signa Aldrich) at 37°C for 60 minutes. Subsequently, cells were treated with Myc primary antibodies (1:100) and hornerin primary antibodies (1:100) at 37°C for 30 minutes. Cells were washed with wash buffer A and treated with PLUS and MINUS PLA probes (1:5) at 37°C for 60 minutes. Cells were washed with wash buffer A and treated with ligase solution at 37°C for 30 minutes. Cells were washed with wash buffer A and treated with polymerase solution at 37°C for 100 minutes. Cells were washed with wash buffer B for 20 minutes and then with 0.01x buffer B for 4 minutes. After the addition of DAPI (4',6-diamidino-2-phenylindole), cells were counted and images were generated.

[0152]

[0153] 13. Centrosome preparation

[0154] Cells were grown in culture medium containing 10 mM nocodazole and 10 mM cytochalasin D at 37°C for 1 h. Cells were harvested and lysed in a solution containing 1 μg / mL protease inhibitor for aprotinin, leupeptin, or pepstatin (1 mM HEPES pH 7.2, 0.5% NP-40, 0.5 mM MgCl2, 0.1% β-mercaptoethanol, 1 mM PMSF). Swollen nuclei and chromatin aggregates were removed by centrifugation at 3,500 rpm for 10 min, and the supernatant was filtered through a nylon mesh. 10 mM HEPES and 1 μg / mL DNase I were added, the lysate was loaded onto a 50% sucrose solution, and centrifuged at 7,500 rpm for 30 min to sediment the centrosomes on a sucrose cushion. One-third of the initial volume of the supernatant was removed, and the resuspended supernatant was loaded onto a sucrose solution that formed a concentration gradient of 3 ml of 70%, 3 ml of 50%, and 3 ml of 40%, and centrifuged at 25,000 rpm for 1 h. Fourteen fractions, each containing 500 μL, were collected from the bottom.

[0155]

[0156] 14. Colony formation analysis

[0157] Cells were seeded at a density of 500 cells per dish and cultured for 2 weeks. The cell culture medium was replaced every 3 days. After 2 weeks, the cells were washed with phosphate-buffered saline (PBS) and stained with crystal violet (0.5 mg / ml, Santa Cruz Biotechnology Inc.) for 30 minutes on a shaker. After 30 minutes, the cells were washed several times with PBS. Colonies were photographed, and those with >50 cells were counted.

[0158]

[0159] 15. TCGA RNA-seq data

[0160] All TCGA RNA-seq data were obtained from the Human Protein Atlas website (www.proteinatlas.org). Patients were divided into two expression groups based on the FPKM (fragments per kilobase of exon per million reads) value of each gene, and the correlation between expression levels and patient survival was examined. Survival outcomes between the two groups were compared using Kaplan-Meier survival analysis and the log-rank test. All statistical analyses were performed using IBM SPSS Statistics version 20.0 (IBM Inc., Armonk, NY, USA). All tests were two-sided, and a p value of 0.05 was used as the cutoff value to determine statistical significance.

[0161]

[0162] Example 1. Evaluation of multipolar spindle formation due to DNA damage

[0163] To discover a new biomarker for predicting or diagnosing cancer cell death through the DNA damage response (DDR), one of the mechanisms of anticancer action, we analyzed whether mitotic catastrophe or death in mitosis (DiM), a phenomenon that specifically occurs when DNA damage occurs in cancer cells, is related to the presence or absence of multipolar spindles. To monitor spindles in HeLa cells, a cervical cancer cell line, HeLa cells were transformed to express GFP-tagged tubulin. To induce DNA damage response (DDR) in the HeLa cells, 0.5 μM benzo[a]pyrene (B[a]P) was treated for 48 h, and then GFP-tubulin was observed for 10 h via live cell imaging to analyze the cell cycle morphology according to the induction of DNA damage response (DDR). For comparison of cell cycle, cells transfected with GFP-tubulin but subjected to the same cell treatment except for B[a]P treatment (Mock) were used as a control.

[0164] As shown in Fig. 2, a total of 1609 cells were observed, and it was confirmed that bipolar spindles were observed in the control group and that mitosis progressed normally over time. On the other hand, in cells (B[a]P) in which the DNA damage response (DDR) was induced, both bipolar spindles and multipolar spindles were observed, and the cell cycle patterns were divided into three types: mitotic cell death (DiM), regression, and apoptosis. The regression type means that cell death did not progress despite treatment with B[a]P (benzo[a]pyrene) and cell division progressed as before treatment with the reagent. In the above results, among the cells (B[a]P) in which the DNA damage response (DDR) was induced, the proportion of cells exhibiting a cell cycle morphology of mitotic cell death (DiM) (n=356) with bipolar or multipolar spindles was analyzed. As shown in Fig. 3, it was confirmed that approximately 70% of the cells progressing into a cell cycle morphology of mitotic cell death (DiM) were cells in which multipolar spindles were observed.

[0165] In addition, among the cells (n=257) in which multipolar spindles were observed in the above results, the proportion of cells that progressed through the cell cycle of apoptosis (DiM) or regression during mitosis was analyzed. As shown in Fig. 4, among the cells in which multipolar spindles were observed, approximately 20% did not undergo apoptosis but rather underwent regression and cell division. Cells that progressed through the cell cycle of regression while exhibiting multipolar spindles produced polylobed progeny.

[0166] The effect of the DNA damage response (DDR) on spindle morphology in cells that had progressed to prometaphase was analyzed. HeLa cells were treated with DNA-damaging agents (DDA), including benzo[a]pyrene (B[a]P), B[a]P-7,8-diol-9,10-epoxide (BPDE), and etoposide. Immunostaining was performed with antibodies that specifically bind to pericentrin and β-tubulin, and DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Mock cells, which were treated with the same treatment except for DDA, were used as controls. In addition, the morphology of the spindle was analyzed in the case of cells with DNA damage caused by radiation by treating the cells with 3 Gy of radiation (IR) instead of treating with a DNA damaging agent (DDA). The pericentrin is a protein that is recruited around the centrosome, and the tendency of centrosome formation was confirmed through the location of the pericentrin within the cell. The β-tubulin is a substance that constitutes the microtubules that form the spindle, and the morphology of the spindle was confirmed through the location of the β-tubulin within the cell.

[0167] As shown in Fig. 5, among the cells that progressed to prometaphase, the control group (Mock) showed a bipolar spindle morphology, but cells treated with a DNA-damaging agent (DDA) showed a multipolar spindle morphology. In addition, cells that were induced to DNA damage by radiation alone without treatment with a DNA-damaging agent (DDA) also showed a multipolar spindle morphology. In addition, among 300 prometaphase cells that were analyzed for spindle morphology, the proportion of cells showing multipolar spindles for each experimental group was analyzed. As shown in Fig. 6, the proportion (%) of cells exhibiting the form of a multipolar spindle was not found in the control group (Mock), was 56% in the B[a]P treatment group, 27% in the BPDE treatment group, 63% in the etoposide treatment group, and 22% in the radiation (IR) treatment group.

[0168] In order to analyze the formation mechanism of multipolar spindles induced by the DNA damage response (DDR) with reference to the above results, cells were treated with NU6027 (2 μM), an ATR inhibitor (ATRi), KU55933 (20 nM), an ATM inhibitor (ATMi), UCN-01 (300 nM), a Chk1 inhibitor (Chk1i), BML-277 (1 μM), a Chk2 inhibitor (Chk2i), or KU57788 (0.5 μM), a DNA-PK inhibitor (DNA-PKi), for 24 hours before treatment with B[a]P and etoposide, which are DNA damaging agents (DDA), and the formation of multipolar spindles was analyzed. As shown in Fig. 7, the results of analysis using live cell imaging showed that multipolar spindles found in cells treated with etoposide did not form multipolar spindles but formed bipolar spindles similar to normal mitosis when pretreated with NU6027, an ATR inhibitor (ATRi), and UCN-01, a Chk1 inhibitor (Chk1i). In addition, the proportion of cells exhibiting multipolar spindles among prometaphase cells in each experimental group was analyzed according to the presence or absence (+ / -) of DNA damaging agent (DDA) treatment. As shown in Figure 8, pretreatment with NU6027, an ATR inhibitor (ATRi), and UCN-01, a Chk1 inhibitor (Chk1i), showed a noticeable decrease in the proportion of cells forming multipolar spindles.

[0169] These results demonstrate that when DNA-damaging agents (DDA) or radiation are administered to cancer cells to induce a DNA damage response (DDR), the proportion of cells that form multipolar spindles and exhibit anticancer effects, such as death in mitosis (DiM), increases. Furthermore, we demonstrate that ATR and Chk1 play a key role in the formation of multipolar spindles in this DDR.

[0170]

[0171] Example 2. Evaluation of the Relationship Between Centriole Duplication and Detachment in DDR and Multipolar Spindle Formation

[0172] Referring to the above results, and noting that multiple centrosomes appear when DDR (DNA damage response) is induced, each DDA (DNA-damaging agent) was treated and the number of formed centrosomes was counted. HeLa cells were treated with DNA-damaging agents (DDA), such as B[a]P((benzo[a]pyrene), BPDE(B[a]P-7,8-diol-9,10-epoxide), or etoposide, and the number of centrioles in prometaphase cells (n=300) was counted by staining with antibodies. As shown in Fig. 9, the number of centrosomes formed by two centrioles was normally 2 in the control group (Mock) as analyzed by immunofluorescence images, 5 in the case of B[a]P((benzo[a]pyrene) treatment, 5 in the case of BPDE(B[a]P-7,8-diol-9,10-epoxide) treatment, and 4 in the case of etoposide treatment. DDR (DNA damage response) Referring to the fact that the number of centrosomes is 4 or more when induced, the proportion of cells with 4 or more centrioles in mitotic cells in each experimental group was analyzed. As shown in Fig. 10, in the case of cells treated with DDA (DNA-damaging agent), the proportion of cells with 4 or more centrioles was found to be more than 70%. Through the above results, it was proven that DDA (DNA-damaging agent) induces centriole overduplication in G2, and ultimately induces centrosome amplification (CA) during mitosis.

[0173] Referring to the above results, considering that ATR and Chk1 play a major role in the process of forming a multipolar spindle, we evaluated whether centriole overduplication is related to the formation of a multipolar spindle. Before treatment with DNA-damaging agents B[a]P and etoposide, cells were treated with NU6027 (2 μM), an ATR inhibitor (ATRi), KU55933 (20 nM), an ATM inhibitor (ATMi), UCN-01 (300 nM), a Chk1 inhibitor (Chk1i), BML-277 (1 μM), a Chk2 inhibitor (Chk2i), or KU57788 (0.5 μM), a DNA-PK inhibitor (DNA-PKi), for 24 h, and the expression levels of Plk4, STIL, and SAS-6, which are key factors in the initiation of centriole replication, were analyzed by immunoblotting and immunostaining.

[0174] As shown in Figures 11 and 12, the expression levels of Plk4, STIL, and SAS-6 were significantly increased in cells treated with DDA (DNA-damaging agent). However, the expression levels of Plk4, STIL, and SAS-6 increased by DDA (DNA-damaging agent) were not affected by the inhibitors. These results demonstrate that centriole overduplication induced by the DNA damage response (DDR) is induced by a separate mechanism from that associated with multipolar spindles.

[0175] As shown in Figure 13, premature centriole disengagement has been reported to occur in cancer cells in early G2 and mitotic phases when treated with anticancer drugs such as doxorubicin. Therefore, we analyzed whether premature centriole disengagement also occurs when the DNA damage response (DDR) is induced over time. HeLa cells expressing GFP-centrin 2 were treated with B[a]P, and centriole positions were analyzed over time using live cell imaging.

[0176] As shown in Figure 14, after treating with DDA and staining with antibodies for centrin 1 and ninein, which are associated with centrosome localization, the distance between each centriole was measured.

[0177] As shown in Figure 15, after treating with DDA and staining with antibodies for centrin 1 and β-tubulin, which are associated with centrosome localization, the intracellular locations of centrin 1 and β-tubulin were confirmed.

[0178] As shown in Figure 16, the proportion of disengaged centrioles in cells that formed multipolar spindles was analyzed.

[0179] The above results demonstrate that inducing DDR in cancer cells results in premature centriole disengagement in the G2 phase, which leads to the formation of multipolar spindles.

[0180] In addition, we evaluated whether the premature centriole disengagement phenomenon was related to the mechanism of multipolar spindle formation. Before treatment with B[a]P, a DNA-damaging agent (DDA), cells were treated with NU6027 (2 μM), an ATR inhibitor (ATRi), KU55933 (20 nM), an ATM inhibitor (ATMi), UCN-01 (300 nM), a Chk1 inhibitor (Chk1i), BML-277 (1 μM), a Chk2 inhibitor (Chk2i), or KU57788 (0.5 μM), a DNA-PK inhibitor (DNA-PKi), for 24 h, and the proportion of cells showing centriole disengagement in prometaphase cells was analyzed. As shown in Figure 17, pretreatment with NU6027, an ATR inhibitor (ATRi), and UCN-01, a Chk1 inhibitor (Chk1i), significantly reduced centriole disengagement induced by DDR.

[0181] These results demonstrate that the formation of multipolar spindles in DDR is associated with premature centriole disengagement rather than centriole overduplication.

[0182] As shown in Figure 18, DDA was treated in non-cancerous RPE-1 (retinal pigment epitilial-1) cells and stained with the indicated antibodies.

[0183] As shown in Figure 19, the proportion of cells with centriole overduplication and premature centriole disengagement was analyzed in non-cancerous RPE-1 (retinal pigment epithelial-1) cells.

[0184]

[0185] Example 3. Analysis of the centriole displacement mechanism in DDR

[0186] Multipolar spindle formation in DDR has been shown to be associated with premature centriole disengagement. Based on these results, we investigated whether changes in the expression of separase, a factor involved in centriole localization, induce premature centriole disengagement when DDR is induced. HeLa cells were transfected with separase siRNA for 24 h, then transfected with a Myc-tagged expression plasmid (Myc), a Myc-separase expression plasmid (WT), or a Myc-tagged noncleavable separase expression plasmid (NC), and then treated with B[a]P for 48 h. Cells were then lysed, and the expression levels of each protein were analyzed using separase and Myc antibodies. HSP90 was used as a control for protein expression levels.

[0187] As shown in Figure 20, endogenous separase was detected in cells that were not transfected with siRNA, but when transfected with siRNA, the expression of endogenous separase was suppressed. In addition, in cells transfected with a plasmid, separase expressed by the plasmid, not endogenous separase, was detected. Considering the above results, we analyzed the change in the proportion of cells showing centriole disengagement according to the presence or absence of siRNA and plasmid infection in prometaphase cells. As shown in Figure 21, there was no significant change in the proportion of cells showing centriole disengagement in prometaphase cells regardless of whether or not they were treated with siRNA and expression plasmid for separase. The above results demonstrate that centriole disengagement induced by DDR is not related to separase.

[0188] Plk1 is an enzyme that phosphorylates the SA / Scc3 cohesin subunit and promotes cohesin dissociation from chromosome arms and centrioles even in the absence of separase activity during prophase. Furthermore, Sgo1 is known to target centrosomes and protect centriole cohesion during early mitosis. Therefore, we assessed whether DDR-induced changes in Plk1 and Sgo1 were associated with premature centriole disengagement. Changes in expression location and levels were assessed by staining cells treated with DDA with Plk1 or Sgo1 antibodies. Expression levels were analyzed relative to centrin expression levels. As shown in Figures 22 and 23, the levels of Plk1 and Sgo1 were significantly reduced in cells treated with DDA. Furthermore, treatment with UCN-01 (300 nM), a Chk1 inhibitor (Chk1i), along with DDA treatment restored the Plk1 level reduced by DDA. These results demonstrate that premature centriole disengagement induced by DDR is associated with reduced levels of Plk1 and Sgo1, and that the DDR-induced decrease in Plk1 expression is mediated by Chk1.

[0189] In addition, as shown in Figure 24, when the expression levels of Plk1 and Sgo1 in the centrosome were analyzed by Western blot, the expression of Plk1 and Sgo1 in the centrosome was found to be reduced in cells treated with DDA, and the phenomenon of Sgo1 translocation to the centrosome due to DDA was found to be drastically reduced.

[0190] As shown in Figures 25 and 26, the interaction between Plk1 and Sgo1 was found to be reduced in cells treated with DDA.

[0191] The above results show that the interaction between Plk1 and Sgo1 is reduced in DDA-treated cells, demonstrating that DDR induces premature centriole disengagement by disrupting the interaction between Plk1 and Sgo1.

[0192]

[0193] Example 4. Effect of Chk1 on the interaction between Plk1 and Sgo1.

[0194] In Example 3 above, it was confirmed that the interaction between Plk1 and Sgo1 was reduced in DDR, and the decrease in Plk1 level by DDA was related to Chk1. Chk1 is known to phosphorylate the polo-box domain (PBD) of Plk1. To investigate the mechanism by which Chk1 prevents Plk1 from moving to the centrosome, the phosphorylation sites in the polo-box domain (PBD) related to the substrate specificity and centrosomal localization of Plk1 were analyzed by immunoprecipitation using mass spectrometry. As a result, among the 14 phosphorylation residues in the polo-box domain (PBD), Ser 526 , Ser 529 and Thr 539were selected as candidates. The changes in DDR-induced multipolar spindles were analyzed by changing the selected phosphorylation sites. HeLa cells were transfected with Myc-tagged (Myc), Myc-tagged wild-type Plk1 (WT), or mutant Plk1 expression plasmids, and the locations of Plk1 and centrin were confirmed using immunoblots. The mutant Plk1 was constructed using a point mutation kit, and Ser of the PBD in Plk1 was 526 , Ser 529 and Thr 539 It was produced as a 3D mutant (S526D / S529D / T539D) in which the phosphorylation-mimicking amino acid was changed to aspartic acid (Asp, D), or as a 3A mutant (S526A / S529A / T539A) in which the phosphorylation-mimicking amino acid was changed to alanine (Ala, A).

[0195] As shown in Figure 27, in cells expressing wild-type Plk1 via a plasmid (WT), Plk1 was localized around the centrosome and formed bipolar spindles. In addition, in cells expressing the 3A mutant Plk1 (3A), which was changed to alanine (Ala, A), which represents non-phosphorylation, Plk1 was also localized around the centrosome and formed bipolar spindles. In contrast, in cells expressing the 3D mutant Plk1 (3D), which was changed to aspartic acid (Asp, D), which mimics phosphorylation, Plk1 was scattered and formed multipolar spindles. The proportion of cells showing centrosome localization in each experimental group was analyzed based on the above results. As shown in Figure 28, in the case of wild-type Plk1 expression (WT) and 3A mutant Plk1 expression (3A) by plasmid, most of the cells showed centrosome localization, whereas in the case of 3D mutant Plk1 expression (3D), the proportion of cells showing centrosome localization was significantly reduced. In addition, the proportion (%) of cells showing multipolar spindles in each experimental group of the above results was analyzed. As shown in Figure 29, the proportion of cells with multipolar spindles increased by treatment with B[a]P, a DDA, was significantly reduced in the case of wild-type Plk1 expression (WT) and 3A mutant Plk1 expression (3A) by plasmid. On the other hand, in the case of 3D mutant Plk1 expression (3D), the proportion of cells with multipolar spindles was increased again. The above results indicate that Ser of PBD in Plk1 526 , Ser 529 and Thr 539It is demonstrated that phosphorylation of is involved in the formation of multipolar spindles within cells.

[0196] To identify the phosphorylation site in endogenous Plk1 and screen the relevant kinase, phospho-Ser 526 , phosphorylated-Ser 529 and phosphorylated-Thr 539 An antibody that specifically binds to was produced. HeLa cells were treated with DDA B[a]P, Cdk1 inhibitor RO3306, or nocodazole, and phospho-Ser in each experimental group was analyzed by immunoblotting. 526 (pS526), ​​phosphorylated-Ser 529 (pS529), phosphorylated-Thr 539 (pT539), Plk1, Chk1, cyclin A, and cyclin B1 levels were analyzed.

[0197] As shown in Figure 30, in cells treated with B[a]P, Ser of Plk1 526 , Ser 529 and Thr 539 Ser phosphorylation was increased in cells treated with RO3306, a Cdk1 inhibitor. 529 and Thr 539 Only phosphorylation was increased, and Ser was not detected in cells treated with nocodazole, an early mitotic inhibitor. 526 Only phosphorylation increased.

[0198] As shown in Figure 31, in G2 arrested cells by RO3306, a Cdk1 inhibitor, Ser 529 and Thr 539 Ser phosphorylation was increased in cells released from G2 arrest 529 and Thr 539 Phosphorylation was shown to decrease.

[0199] The above results indicate that Ser of Plk1 during G2 and mitosis 526 , Ser 529and Thr 539 It demonstrates that the phosphorylation of each of them functions independently.

[0200] Referring to the above results showing that the level of Plk1 in the centrosome was restored by Chk1 inhibitors, we evaluated whether each inhibitor also affected the phosphorylation of Plk1. As shown in Figure 32, when treated with ATR and Chk1 inhibitors, Ser induced by [a]P treatment 529 and Thr 539 It was shown to eliminate phosphorylation of Ser 526 It was found that it did not affect phosphorylation.

[0201] In addition, we evaluated which site of phosphorylation of Chk1 in Plk1 is involved in Thr 210 , Ser 526 , Ser 529 or Thr 539 HeLa cells were transfected with plasmids expressing point mutations T210A, S526A, S529A, and T539A, which were changed to alanine (Ala, A) representing nonphosphorylation, to express His-Plk1, and then cultured with Chk1, and the protein levels of each were analyzed by immunoblotting. As shown in Figure 33, Chk1 was expressed at Thr of Plk1. 210 In addition to phosphorylation, Ser 529 or Thr 539 It was shown to directly phosphorylate .

[0202] To exclude the possibility of autophosphorylation by active Plk1, Chk1 was mutated to Ser in arginine mutated Plk1 (K82R) at dominant-negative lysine 82. 529 or Thr 539 As shown in Figure 34, even in the K82R mutant that has lost autophosphorylation functionality, Ser529 or Thr 539 Phosphorylation of Plk1 was confirmed. We also evaluated whether Chk1 interacts with Plk1 in vitro. Recombinant Chk1 was incubated with recombinant His-Plk1, and the protein was pulled down with Ni beads and analyzed by immunoblotting. As shown in Figure 35, it was confirmed that Chk1 directly interacts with Plk1. The results indicate that in DDR, Chk1 binds to Ser in the PBD of Plk1. 529 and Thr 539 It is demonstrated that it is a kinase that phosphorylates .

[0203] Meanwhile, Chk1 inhibitors have been shown to inhibit Ser 526 Nocodazole does not affect the phosphorylation of Ser 526 In view of the above results that increased phosphorylation of Ser, we evaluated whether there were other kinases involved in phosphorylation other than Chk1 in mitosis. Cells were treated with B[a]P or Nocodazole, and treated with RO3306, a Cdk1 inhibitor, BI2536, a Plk1 inhibitor, VX680, an Aurora A inhibitor, or Hesperadin, an Aurora B inhibitor, and Ser 526 The change in phosphorylation level (pS526) was analyzed. As shown in Fig. 36, Ser was only detected when VX680, an Aurora A inhibitor, was treated. 526 The phosphorylation level (pS526) of Plk1 was found to be significantly reduced. Referring to the above results, in vitro, Aurora A was also found to be significantly reduced in Ser 526 To determine whether the phosphorylation of Thr of Plk1 occurs, recombinant His-Plk1 and Aurora A were co-incubated, pulled down with Ni beads, and analyzed by immunoblotting. As shown in Figure 37, Aurora A inhibits the phosphorylation of Thr of Plk1 during late G2 and early mitosis. 210In addition to phosphorylation, Ser of Plk1 526 It was confirmed that phosphorylation occurs. The above results show that Aurora A is a Thr of Plk1 during mitosis. 210 In addition to activation of Plk1 through phosphorylation, Ser of Plk1 is also involved in mitosis with DNA damage. 526 It is demonstrated that it is involved in mitotic catastrophe through phosphorylation.

[0204] To determine whether phosphorylation of Plk1 affects its interaction with Sgo1, we performed an in vitro assay. Cell lysates were obtained from cells transfected with Plk1 wild-type (WT), Plk1 3A mutant, and Plk1 3D mutant, incubated with recombinant His-Sgo1, and the proteins were pulled down with Ni beads and analyzed by immunoblotting. As shown in Figure 38, the Plk1 3D mutant showed a marked decrease in its interaction with Sgo1. These results demonstrate that Chk1-induced phosphorylation of Plk1 in DDR inhibits the interaction between Plk1 and Sgo1, and inhibits the translocation of Plk1 and Sgo1 to the centrosome.

[0205]

[0206] Example 5. Effect of hornerin on phosphorylation of Plk1 by Chk1

[0207] Given that Chk1 is involved in DDR-induced Plk1 phosphorylation, hornerin was identified as a mediator involved in the binding of Chk1 and Plk1 by mass spectrometry analysis of the mediator between Chk1 and Plk1 through purification of the Plk1 complex. In hornerin deletion mutants, Chk1 and Plk1 were confirmed to bind to the N-terminus and C-terminus of hornerin, respectively, and hornerin was confirmed to form a complex with Chk1 and Plk1 in DDR. Therefore, we evaluated whether the presence or absence of hornerin affects the interaction between Chk1 and Plk1 in DDR. HeLa cells were transfected with hornerin siRAN type A and type B, and after treatment with B[a]P, the changes in the interaction between Chk1 and Plk1 were analyzed by IP-immunoblotting, immunostaining, and immunoblotting.

[0208] As shown in Figure 39, the interaction between Chk1 and Plk1 was completely inhibited in cells depleted of hornerin by siRAN. Furthermore, as shown in Figures 40 and 41, the levels of Plk1 and Sgo1 at the centrosome, which were reduced due to DDR, were restored in cells depleted of hornerin by siRAN. These results demonstrate that hornerin affects the inhibition of the translocation of Plk1 and Sgo1 to the centrosome due to the interaction between Chk1 and Plk1, and that depletion of hornerin inhibits the interaction between Chk1 and Plk1, thereby promoting the translocation of Plk1 and Sgo1 to the centrosome, thereby inducing normal formation of a bipolar spindle.

[0209] To specifically evaluate whether the above hornerin affects the phosphorylation of Plk1 by Chk1, the Thr of Plk1 following depletion of hornerin in DDR 210 , Ser 526 , Ser 529 or Thr 539 The phosphorylation level of hornerin was analyzed. As shown in Figure 42, depletion of hornerin in DDR 529 and Thr 539 It was shown to significantly reduce phosphorylation of Thr 210 and Ser 526 It was found that the depletion of hornerin did not affect the phosphorylation of Plk1. That is, the above results suggest that the depletion of hornerin affects Ser of Plk1. 529 and Thr 539 By significantly reducing the phosphorylation of hornerin, the interaction between Chk1 and Plk1 is inhibited, and the translocation of Plk1 and Sgo1 to the centrosome progresses, demonstrating normal formation of a bipolar spindle. Meanwhile, as shown in Figure 43, depletion of hornerin did not dissociate the Cep192::Aurora A::Plk1 complex. These results confirmed that even when the DNA damage response (DDR) was induced in hornerin-depleted cells, the Cep192::Aurora A::Plk1 complex did not dissociate, but rather translocated to the centrosome, preventing centriole disengagement.

[0210] Referring to the above results showing that phosphorylation of Plk1 is related to hornerin, the interaction with hornerin depending on the phosphorylation status of Plk1 was evaluated. HeLa cells were infected with plasmids expressing recombinant Plk1 wild type (WT), 3A mutant (3A), or 3D mutant (3D). For in vitro experiments, cell lysates were obtained from infected HeLa cells, incubated with recombinant His-hornerin, pulled down with Ni beads, and analyzed by immunoblotting. As shown in Figure 44, Ser in vitro 526 , Ser 529 or Thr 539 The 3D mutant representing phosphorylated Plk1 was shown to interact strongly with hornerin. In addition, IP-immunoblotting was performed with HeLa cells. As shown in Figure 45, Ser 526 , Ser 529 or Thr 539 In the 3D mutant representing phosphorylated Plk1, hornerin was found to interact strongly with Cep192. In addition, proximity ligation assays were performed in the infected HeLa cells. As shown in Fig. 46, in situ Ser 526 , Ser 529 or Thr 539 Hornerin was found to interact strongly with cells infected with a plasmid expressing a 3D mutant representing phosphorylated Plk1.

[0211] To assess whether hornerin localizes according to the cell cycle, hornerin was immunostained and its location was determined as the cell cycle progresses. As shown in Figure 47, hornerin was not localized to the centrosome but was scattered throughout the cytoplasm, depending on the cell cycle stage: prophase, prometaphase, metaphase, early anaphase, late anaphase, and telophase.

[0212] In summary, these results demonstrate that even when a DNA damage response (DDR) is induced in cells, the absence of hornerin prevents the formation of multipolar spindles, prevents death in mitosis (DiM), and allows cells to divide with damaged DNA. These findings suggest that hornerin may serve as a prognostic marker for anticancer treatments that induce a DNA damage response (DDR), such as DDA-based anticancer drugs or radiation.

[0213]

[0214] Example 6. Effect of DDR on structural changes and activation of Plk1.

[0215] Referring to the above results that hornerin is related to the phosphorylation of Plk1 and that depletion of hornerin prevents the formation of multipolar spindles and the initiation of death in mitosis (DiM), Ser 526 , Ser 529 and Thr 539We also evaluated whether hornerin depletion had an effect on the Plk1 3D mutant, which represents phosphorylated Plk1. HeLa cells (n=300) were infected with siRNA for 24 h to remove hornerin, and then transfected with Plk1 wild type (WT), Plk1 3A mutant, or Plk1 3D mutant. DDR was induced, and immunohistochemistry was used to analyze whether Plk1 translocated to the centrosome. As shown in Figure 48, in cells transfected with Plk1 wild type (WT) or Plk1 3A mutant, hornerin depletion resulted in an increase in Plk1 at the centrosome, whereas in cells transfected with Plk1 3D mutant, hornerin depletion did not affect the decrease in Plk1 at the centrosome.

[0216] Based on the above results showing that depletion of hornerin does not dissociate the Cep192-Aurora A-Plk1 complex and translocates it to the centrosome, preventing centriole disengagement, we assessed whether DDR affects the translocation of Cep192 and Aurora A to the centrosome. Cep192 is known to recruit Plk1 and Aurora A to the centrosome, and the kinase activity of Plk1 plays a pivotal role in targeting it to the centrosome, and hornerin and Chk1-mediated activation of the PBD region of Plk1 Ser 526 , Ser 529 and Thr 539It was confirmed whether phosphorylation of can affect the activity of Cep192. As shown in Figures 49 and 50, the levels of Cep192 and Aurora A at the centrosome were significantly reduced in cells in which DDR was induced by B[a]P. In addition, after transfection with Plk1 wild type (WT), Plk1 3A mutant, and Plk1 3D mutant, DDR was induced, and immunostaining was used to analyze whether Cep192 was translocated to the centrosome. As shown in Figure 51, the level of Cep192 at the centrosome was maintained in cells transfected with Plk1 wild type (WT) or Plk1 3A mutant, but the level of Cep192 at the centrosome was significantly reduced in cells transfected with Plk1 3D mutant. Note that the Plk1 3D mutant strongly interacts with Cep192, Ser 526 , Ser 529 and Thr 539 Phosphorylated Plk1 was confirmed to inhibit the translocation of Cep192 to the centrosome through binding to Cep192. Meanwhile, whether phosphorylated Plk1 affects the centrosome translocation of Sgo1 was evaluated using the same method as above. As shown in Figure 52, in contrast to the results for Cep192, the level of Sgo1 at the centrosome was not significantly changed in cells transfected with Plk1 wild type (WT), Plk1 3A mutant, or Plk1 3D mutant. The results confirm that even if mutant phosphorylated Plk1 injected into the plasmid is present in the cell, endogenous Plk1 does not cause Sgo1 to translocate to the centrosome to form a multipolar spindle.

[0217] Plk1 undergoes structural changes upon phosphorylation, and Thr of Plk1 in the T-loop 210 Phosphorylation is the activated form of Plk1, and Thr of Plk1 210When phosphorylated, Plk1 cannot undergo conformational changes in the T-loop. Ser in the PBD region of Plk1 526 , Ser 529 and Thr 539 Although phosphorylation of Thr is not localized to the centrosome, we analyzed the principle that depletion of hornerin in Plk1 3D mutants does not affect the inhibition of centrosome translocation due to phosphorylation of Plk1. 210 T210A mutation representing phosphorylation, PBD region Ser of Plk1 526 , Ser 529 and Thr 539 3D mutations (S526D / S529D / T539D) representing phosphorylation of Plk1, and Thr 210 and PBD region Ser 526 , Ser 529 and Thr 539 Plasmids expressing 4D mutations (T210D / S526D / S529D / T539D) representing phosphorylation were constructed, and it was confirmed whether Plk1 was localized to the centrosome in cells expressing each mutation. As shown in Figure 53, in the T210A mutation and the 4D mutation, about half of the Plk1 was localized to the centrosome as in the WT. In addition, cells expressing each mutation were treated with B[a]P to induce DDR, and the proportion of prometaphase cells showing multipolar spindles was analyzed. As shown in Figure 54, in the T210A mutation and the 4D mutation, the proportion of prometaphase cells showing multipolar spindles was significantly reduced, similar to the WT. On the other hand, in the Plk1 3D mutation, Thr 210 To determine whether phosphorylation was present, cell lysates were obtained from cells infected with the Plk1 3D mutant, and Thr 210 Using antibodies specific for phosphorylation of Thr 210The presence of phosphorylation (pPlk1(T210)) was analyzed. As shown in Figure 55, in the Plk1 3D mutant, Thr 210 No phosphorylation was detected. The results indicate that the PBD site Ser of Plk1 is induced by DDR. 526 , Ser 529 and Thr 539 As phosphorylation progresses, conformational changes in Plk1 and Thr 210 We demonstrate that phosphorylation is inhibited, rendering Plk1 completely inactive.

[0218] In summary, in DDR-induced cells, hornerin-mediated PBD region Ser of Plk1 526 , Ser 529 and Thr 539 We demonstrate that Plk1 phosphorylation induces the phosphorylation of Cep192::Aurora A::Plk1 complex and Sgo1, and that Plk1 with phosphorylated PBD region maintains a conformational change and inactive state, thereby preventing the translocation of Cep192::Aurora A::Plk1 complex and Sgo1 to the centrosome, thereby inducing death in mitosis (DiM) with multipolar spindle.

[0219]

[0220] Example 7. Effect of hornerin on DDR-induced mitotic cell death.

[0221] The effect of hornerin on the cell cycle changes induced by DDR was evaluated. HeLa cells were infected with siRNA to remove hornerin, and then treated with B[a]P or etoposide. Colony formation was analyzed for 14 days, and the size and number of each colony formed were measured. As shown in Figure 57, depletion of hornerin was found to significantly reduce the size of the colonies. On the other hand, as shown in Figures 56 and 58, depletion of hornerin increased the number of colonies, and cell viability was found to increase by more than 50% even when DDR was induced by treatment with B[a]P or etoposide. In addition, depletion of hornerin was found to induce a phospholipid-binding domain (PBD) of Plk1, Ser 526 , Ser 529 and Thr 539 Not only phosphorylation of Ser of Chk1 but also 345 We evaluated whether it affects phosphorylation. HeLa cells were infected with siRNA to remove hornerin, and then treated with B[a]P or etoposide. Using immunoblotting, the PBD region Ser of Plk1 was analyzed. 526 , Ser 529 and Thr 539 Phosphorylation of and Ser of Chk1 345 The phosphorylation level was analyzed. As shown in Figure 59, depletion of hornerin resulted in a decrease in the PBD region Ser of Plk1. 529 and Thr 539 , but the PBD region Ser of Plk1 affected the phosphorylation 526 Phosphorylation and Ser of Chk1 345 It was shown that it had no effect on phosphorylation.

[0222] To assess whether the prognosis of various cancer patient groups is related to the expression level of Hornerin, we analyzed the changes in the survival rate of patients with cervical, pancreatic, or renal cancer according to the mRNA expression level of Hornerin in the TCGA database. As shown in Figure 60, when the expression level of Hornerin was high, 50% of patients survived over time. However, when the expression level of Hornerin was low, the survival rate of patients decreased sharply, and all patients died by 6 years. These results demonstrate that the expression level of Hornerin can serve as a marker for predicting the prognosis of cancer patients.

[0223]

[0224] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for predicting the prognosis of cancer patients, comprising hornerin.

2. A biomarker composition according to claim 1, characterized in that the cancer patient has received or will receive anticancer treatment using a DNA damage response.

3. A biomarker composition according to claim 2, wherein the anticancer treatment is anticancer drug treatment or radiation therapy.

4. In the first paragraph, the biomarker composition comprises Plk1 in which the 526th serine from the N-terminus in the amino acid sequence represented by SEQ ID NO: 2 is phosphorylated; Plk1 phosphorylated at serine 529 from the N-terminus in the amino acid sequence represented by sequence number 2; Plk1 phosphorylated at the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2; Plk1 in which the 529th serine and the 539th threonine from the N-terminus are phosphorylated in the amino acid sequence represented by sequence number 2; or A biomarker composition characterized in that it further comprises Plk1; wherein the 526th serine, the 529th serine, and the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2 are phosphorylated.

5. A biomarker composition for screening suitability for anticancer treatment comprising hornerin.

6. A biomarker composition for screening suitability for anticancer treatment, characterized in that the anticancer treatment in paragraph 5 is anticancer drug treatment or radiation therapy utilizing DNA damage response.

7. A kit for screening suitability for anticancer treatment, comprising a preparation for measuring the expression level of Hornerin.

8. A kit for screening suitability for anticancer treatment, characterized in that in paragraph 7, the agent for measuring the expression level is a primer or probe that specifically binds to a gene encoding Hornerin, or an antibody, peptide, aptamer, or compound that specifically binds to Hornerin.

9. In the 7th paragraph, the kit for screening suitability for anticancer treatment further comprises a Plk1 phosphorylation measuring agent that measures the phosphorylation level of the 526th serine, the 529th serine, or the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2. A kit for screening suitability for anticancer treatment.

10. A kit for screening suitability for anticancer treatment, characterized in that in paragraph 9, the agent for measuring phosphorylation of Plk1 is an antibody, peptide, aptamer or compound that specifically binds to phosphorylation of the 526th serine, the 529th serine or the 539th threonine from the N-terminus in the amino acid sequence represented by sequence number 2.

11. A step of measuring the mRNA expression level or protein expression level of Hornerin in a biological sample isolated from a cancer patient; and A method for providing information for predicting the prognosis of a cancer patient, comprising: a step of predicting that the patient's prognosis will be good if the mRNA expression level or protein expression level of the hornerin is higher than that of a control sample; 12. In the 11th paragraph, the method for providing information for predicting the prognosis of a cancer patient comprises the steps of: measuring whether the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 consisting of the amino acid sequence represented by sequence number 2 is phosphorylated in a biological sample isolated from a cancer patient; and A method for providing information for predicting the prognosis of a cancer patient, further comprising: a step of predicting that the patient's prognosis will be good when phosphorylation of the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 consisting of the amino acid sequence represented by the above sequence number 2 is detected; 13. A method for providing information for predicting the prognosis of a cancer patient, characterized in that the cancer patient in paragraph 11 is a patient who has received or will receive anticancer treatment using a DNA damage response.

14. A method for providing information for predicting the prognosis of a cancer patient, characterized in that the biological sample is tumor tissue in the 11th paragraph.

15. A method for providing information for determining suitability for anticancer treatment, comprising the step of measuring the level of hornerin in a biological sample isolated from a cancer patient prior to receiving anticancer treatment.

16. A method for providing information for determining the suitability of anticancer treatment, characterized in that the anticancer treatment in paragraph 15 is anticancer treatment or radiation therapy utilizing DNA damage response.

17. A method for providing information for determining the suitability of anticancer treatment, characterized in that the biological sample is tumor tissue in the 15th paragraph.

18. A method for providing information for determining the suitability of anticancer treatment, characterized in that, in the 15th paragraph, if hornerin is detected in the biological sample, anticancer treatment is determined to be suitable.

19. In the 15th paragraph, the method for providing information for determining the suitability of the anticancer treatment further comprises a step of measuring whether the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 consisting of the amino acid sequence represented by sequence number 2 is phosphorylated in a biological sample isolated from a cancer patient.

20. A method for providing information for determining the suitability of anticancer treatment, characterized in that, in paragraph 19, if phosphorylation of the 526th serine, the 529th serine, or the 539th threonine from the N-terminus of Plk1 is detected in the biological sample, the anticancer treatment is determined to be suitable.

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