KR101096928B1 - Biomarker for the diagnosis of anti-cancer medicine resistance of breast cancer patients and kit - Google Patents

Abstract

The present invention relates to a biomarker and kit for diagnosing chemotherapy resistance in breast cancer patients for performing selective preoperative chemotherapy for breast cancer patients. The present invention relates to a biomarker for diagnosing chemotherapy resistance in breast cancer patients and to anticancer drug resistance in breast cancer patients. In order to provide the necessary information, a method for measuring the degree of phosphorylation of the biomarker protein and a kit for diagnosing anticancer drug resistance in a breast cancer patient are provided. The present invention enables early prediction of anticancer drug resistance of breast cancer patients, thereby enabling selective and individual use of anticancer drugs for breast cancer patients, thereby improving the quality of life of breast cancer patients and inducing rational use of anticancer drugs related medical expenses. It is expected to be.

Breast cancer, chemotherapy, biomarkers

Description

Biomarker for the diagnosis of anti-cancer medicine resistance of breast cancer patients and kit}

The present invention relates to a biomarker and kit for diagnosing chemotherapy resistance in breast cancer patients for performing selective preoperative chemotherapy for breast cancer patients, and more particularly, to a biomarker for diagnosing chemotherapy resistance in breast cancer patients, and to anticancer drug resistance in breast cancer patients. The present invention relates to a method for measuring the degree of phosphorylation of the biomarker protein and a kit for diagnosing anticancer drug resistance in breast cancer patients to provide information for diagnosis.

Preoperative chemotherapy for breast cancer patients was first attempted in the 1970s, and its effectiveness has been demonstrated in many studies. Recently, postoperative chemotherapy and preoperative chemotherapy have been shown to improve patient survival. As it turns out, the utility is further emphasized.

However, chemotherapy is generally a costly and expensive treatment that causes toxic side effects in the human body and is economically expensive. In particular, since only a small number of patients improve the survival rate by the actual chemotherapy, efforts have recently been made to select a patient group that responds to the chemotherapy in terms of personalized therapy. Mammaprint ^® or OncotypeDx ^® , commercially available kits using prognostic factors of breast cancer patients, is a representative result of a study to select a group of patients with early prognosis who have poor prognosis requiring chemotherapy.

In recent years, preoperative chemotherapy has attracted much attention as a platform for basic research related to drug development and chemotherapy resistance as well as improving quality of life. Pathologic Complete Response (pCR) after preoperative chemotherapy in the NSABP B-18 study has been reported to be the only predictor of the patient's prognosis. PCR is used as a surrogate marker to confirm the effectiveness of a new treatment in a short period of time after preoperative chemotherapy.

In addition, to identify required resistance related markers or intrinsic resistance related markers inherent in tumors that can confirm the effectiveness of preoperative cancer chemotherapy at an earlier time. Various attempts have been made, and most of these studies are done on preoperative chemotherapy platforms.

 In recent years, the use of preoperative anticancer chemotherapy using functional molecular imaging techniques is underway to attempt to predict the change of tumor cells during the use of anticancer drugs, the prediction of pathologic complete remission, and the progress of tumor response before surgery. have. Previous clinical images compared anatomical changes focused on changes in tumor size, but recent magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MR), photon emission tomography (PET), and single photon emission (SPECT) computed tomography takes advantage of functional changes at the molecular level.

 Toxic side effects of anticancer drugs vary depending on individual and ethnic characteristics, and may be life threatening depending on the therapeutic dose used. The toxicity and side effects should be carefully observed and managed when using anticancer drugs. For this reason, many researches on patient prognostic factors have been conducted, and based on these studies, it was attempted to determine the selective administration of anticancer drugs.

As a study related to the biological characteristics of individual cancers and the sensitivity of anticancer agents, the sensitivity test of anticancer agents in gastric cancer, lung cancer and colon cancer using cell lines has been studied in Korea. Recently, MTS ((3,-( Expression of p53 and bcl-2 proteins, which are related to apoptosis, using the 4,5-dimethylthiazol-2yl) -5- (3-carboxy-methoxyphenyl) -2 (4-sulfophenyl) -2H-tetrazolium, inner salt) method A comparative study of susceptibility test results reported that bcl-2 protein expression was associated with anticancer drug resistance, regardless of p53 expression.

As a result of overseas studies, as a test for drug susceptibility in Japan, a report was made using selective separation of cancer cells from cancer tissues and MTT analysis method, and in the United States, various methods used for drug susceptibility testing in gynecological tumors and breast cancer were reviewed. Announced.

Efforts have been made to develop functional molecular imaging techniques and blood and tissue predictive markers. However, there have been no satisfactory predictors, and development thereof has been required.

The present invention is to overcome the limitations of the prior art as described above, to provide a biomarker that can predict the anti-cancer drug resistance early by using a sample of the breast cancer tissue of the patient, to provide information necessary for diagnosing the anti-cancer drug resistance of the breast cancer patient, An object of the present invention is to provide a method for measuring the degree of phosphorylation of a biomarker protein and a kit for diagnosing anticancer drug resistance in breast cancer patients.

In order to achieve the above object, the present invention provides a biomarker for diagnosing anticancer drug resistance in a breast cancer patient having an amino acid sequence as shown in SEQ ID NO: 1.

The biomarker is a protein named p90RSK, and is composed of a total of 750 amino acids as specified in SEQ ID NO: 1. The present inventors found that when doxorubicin is activated in breast cancer cell lines MDA-MB-231 and BT474, phosphorylation of p90RSK including activation of p42 / p44 MAPK is accompanied (Small GW et al, Pharmacol). Exp Ther , 2003 ), a study was conducted to identify changes in the expression of p90RSK and phospho-p90RSK in breast cancer cell lines other than the two cell lines described above. In the study according to the present invention, rather than simply based on the total amount of the candidate substance, the study proceeds by measuring the expression ratio of p90RSK and phospho-p90RSK in order to link the ratio of the phosphorylated form, which is the active form, to the response of the anticancer agent. It was. As a result, when the expression rate of phospho-p90RSK was compared with the p90RSK expression rate, it was found that the resistance to anticancer drugs decreased as the relative increase was higher, and thus, p90RSK could be used as a biomarker for diagnosing cancer drug resistance in breast cancer patients. I found it possible.

The present invention includes an antibody specifically binding to a biomarker protein of SEQ ID NO: 1 or an immunogenic fragment thereof and an antibody specifically binding to a phosphorylated biomarker protein of SEQ ID NO: 1 or an immunogenic fragment thereof The present invention provides a kit for diagnosing anticancer drug resistance.

Kits of the present invention comprises a secondary antibody conjugate conjugated to a label that is developed by reaction with a substrate; A color substrate solution to be colored and reacted with the label; Washing liquid; And an enzymatic stop solution.

The marker of the secondary antibody conjugate in the kit of the present invention may be selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase, colloidal gold, fluorescents and dyes. Can be.

The color substrate in the kit is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine) It may be selected from the group consisting of.

In another aspect, the present invention provides a method for measuring the degree of phosphorylation of the biomarker protein specified in SEQ ID NO: 1 by using an antigen-antibody binding reaction from the sample in order to provide information necessary for the diagnosis of cancer drug resistance in breast cancer patients.

Method for measuring the degree of phosphorylation of the biomarker protein specified in SEQ ID NO: 1 of the present invention comprises the steps of 1) coating the protein of the sample and the control to the fixture; 2) adding an antibody specifically binding to a biomarker protein of SEQ ID NO: 1 or an immunogenic fragment thereof and a phosphorylated antibody specifically binding to a biomarker protein of SEQ ID NO: 1 or an immunogenic fragment thereof Performing an antigen-antibody binding reaction; 3) detecting the antigen-antibody binding reactant produced through the antigen-antibody binding reaction using a secondary antibody conjugate and a color substrate solution; And 4) comparing the detection results for the sample and the control.

In the method for measuring the phosphorylation degree of the biomarker protein of SEQ ID NO: 1 of the present invention, the sample of step 1) may use tissue isolated from a breast cancer patient, the fixture of step 1) is nitro It can be selected from the group consisting of cellulose membranes, PVDF membranes, well plates synthesized from polyvinyl or polystyrene resins and slide glass of glass.

In the method for measuring the phosphorylation degree of the biomarker protein of SEQ ID NO: 1 of the present invention, the fixture of step 1) is a nitrocellulose membrane, PVDF membrane, polyvinyl (polyvinyl) or polystyrene (polystyrene) resin It may be selected from the group consisting of synthesized well plates and glass slide glass.

In the method for measuring the degree of phosphorylation of the biomarker protein of SEQ ID NO: 1 of the present invention, the antigen-antibody binding reaction of step 2) is enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (radioimmunoassay, RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation.

In the method for measuring the degree of phosphorylation of the biomarker protein of SEQ ID NO: 1 of the present invention, the label of the secondary antibody conjugate of step 3) is composed of HRP, basic dephosphatase, colloidal gold, fluorescent material and pigment Can be selected from the group.

In the method for measuring the phosphorylation degree of the biomarker protein of SEQ ID NO: 1 of the present invention, the color substrate of step 3) may be selected from the group consisting of TMB, ABTS and OPD.

The types of anticancer agents used in chemotherapy include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and monoclonal antibodies. ), And some other anticancer drugs.

Among the anticancer drugs, anthracycline (anthracycline) is used for treating a wide range of cancers and alleviating symptoms. Anthracycline-based anticancer drugs have a mechanism of fundamentally blocking cell division by damaging DNA / RNA base pair strands of cells, and when used, it is known that more active cells, such as cancer cells, become more damaged. These anticancer drugs include a compound called daunosamine, and representative anticancer drugs include daunorubicin, idarubicin, mitomycin, doxorubicin, epirubicin, bleomycin, and dactinomycin. Tinomycin D).

Anticancer drug resistance diagnostic markers and kits for breast cancer patients according to the present invention may be preferably used for anthracycline-based anticancer agents, more preferably for doxorubicin.

The biomarker for diagnosing anticancer drug resistance in breast cancer patients according to the present invention, the method for measuring the degree of phosphorylation of the biomarker protein, and the kit for diagnosing anticancer drug resistance in breast cancer patients for providing information necessary for diagnosing anticancer drug resistance in breast cancer patients are anticancer drug resistance in breast cancer patients. It is expected to enable early and predictive treatment, thereby enabling selective and individual chemotherapy for breast cancer patients, and further improving the quality of life of breast cancer patients as well as the rational use of anti-cancer drug costs.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the present invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to explain in detail the present invention to those skilled in the art.

Example  1: Bio of SEQ ID NO: 1 in breast cancer cell lines Marker  Confirmation of Protein Presence

Breast cancer cell lines (MCF7, MCF7-s, MDA-MB-231, MDA-MB-231s, MDA-MB-436, MDA-MB-468, ZR-75-1, BT474, HS578T, T47D) and the control MCF- Proteins were extracted and purified at 10A (breast cell line) and separated on gels with a 12.5% gradient. The isolated protein was transferred to a nitrocellulose membrane in a solution containing 50 mM Tris, 130 mM glycine, 0.05% SDS, 12.5% methanol. TBST (Tris buffered saline tween 20) containing 5% skim milk powder was added to the transferred membrane to block nonspecific binding, and the primary antibodies anti-p90RSK and anti-phospho p90RSK were diluted 1: 1500 in TBST for 2 hours. Reacted with the membrane during the process. Subsequently, the secondary antibody anti-rabbit IgG-HRP diluted to 1: 5000 was reacted for 1 hour, and ECL (Enhanced Chemiluminance) was added again to the X-ray film in the dark.

These results are shown in "FIG. 1A". Total p90RSK refers to the entirety of p90RSK including both phosphorylated p90RSK and non-phosphorylated p90RSK, and Phospho-p90RSK refers to only phosphorylated p90RSK. Tublin-alpha and NC membranes are shown as Control.

The expression levels of p90RSK and phospho-p90RSK of each cell line of FIG. 1A were measured and quantified using an optical densitometer (FIG. 1B), and then phosphorylated compared to total (Phospho). The expression ratio of was shown in "FIG. 1C" by log substitution.

As such, it was confirmed that the p90RSK protein was present in the cell line in the phosphorylated form and the non-phosphorylated form. From FIG. 1C, the entire p90RSK (including both phosphorylated and non-phosphorylated forms) and phosphorylated p90RSK were identified. The expression rate of could be derived.

Example  2: breast cancer cell line To doxorubicin  Susceptibility measurement and p90RSK (phospho type / total Comparison with the rate of expression

Count 2 X 10 ⁵ cells in each cell line in a 60mm TC dish and treat it for 72 hours with 10 nM of doxorubicin in a conflient state, drop the cells with trypsin, and centrifuge at 1200 rpm. Add 1 ml of medium to the collected pellets and pipette, and then extract 200 ul and place it in a tube containing 600 ul of 1% trypsin and 200 ul of 0.4% trypan blue. Pipette again and pipet 9 ul. Only live cells (N) that were not stained with trypsin blue were counted in a hemocytometer. The experimental group was not treated with doxorubicin. Each cell line was treated with doxorubicin to measure the sensitivity, and the results shown in FIG. 2A are shown.

Cell concentration (Cell / ml) = N × 10 ⁴

As shown in Figure 2a, each cell line showed a variety of reactivity to doxorubicin, which is shown in a graph as shown in Figure 2b below.

The expression ratio of p90RSK (phospho type / total type) is shown in FIG. 1C showing the logarithmic substitution value and FIG.

Expression of p90RSK (phospho type / total type)
Logarithmic substitution of the ratio; Figure 1c
* The higher the value, the higher the phosphorylation rate Sensitivity to doxorubicin; Figure 2b
* The lower the value, the lower the tolerance MCF 7 0.69 3% MCF 7-S -0.06 52% MDA-MB-231 0.71 2% MDA-MB-231-S 0.26 18% MDA-MB-436 1.00 5% MDA-MB-468 0.08 0% ZR-75-1 -0.65 65% BT474 0.27 18% HS578T -0.93 80% T47D 0.53 32% MCF-10A1
(control) 0.69 50%

In the table, MCF7, MDA-MB-231, MDA-MB-231-s, MDA-MB-436, MDA-MB-468, BT474, and T47D, which have a relatively high proportion of phosphorylated p90RSK, are treated with doxorubicin. The percentage of living cells before and after treatment was 3%, 2%, 18%, 5%, 0%, 18% and 32%, respectively, with high susceptibility to doxorubicin (low resistance to anticancer drugs). MCF7-s, ZR-75-1, and HS578T, which had relatively low p90RSK ratios, showed 52%, 65%, and 80% viable cell counts after treatment with doxorubicin. Low susceptibility to doxorubicin (high resistance to anticancer drugs). From this, it was found that the cell line having a low phosphorylation rate of p90RSK increased doxorubicin resistance.

Example  3: p90RSK Correlation with Acquisition of Anticancer Drug Resistance in Cell Lines in Relation to the Mechanism of Action

Using the ingenuity pathway analysis system, a database that can analyze molecular biological protein pathways, analyzes the relationship between p90RSK and mitogen-activated protein kinase (MAPK) mechanisms involved in anticancer activity. The degree of mechanism activity was analyzed through several representative protein bodies (FIGS. 3A and 3B).

For "Fig. 3b," and "Fig. 2b" for comparison, the MDA-MB-231 in the mechanism of action p90RSK is actively enabled and MDA-MB-436 has been found that the sensitivity to doxorubicin very high. Therefore, the smooth signaling of a series of proteins involved in the mechanism of action of p90RSK could suggest the possibility of anticancer action.

Example  4: excavated Biomarker  Obtain clinical samples for ideal validation Clinical validation set  build)

 In order to confirm the results in the cell line using a human sample, the central acupuncture biopsy tissue was collected and stored before the start of chemotherapy before surgery, and the tissue of the patient was collected and stored at the time of mastectomy after chemotherapy for a certain period. It was. A total of 10 patients were treated with tissue before and after chemotherapy. The response to chemotherapy was assessed by Response Evaluation Criteria in Solid Tumor (RECIST), and the relevant clinicopathological factors were collected.

Specific evaluation methods are as follows. After breast cancer tissue before chemotherapy, the patient was observed for chemo reactivity, and then divided into resistance and response groups according to the degree of reactivity, and then 5 cases (resistance group; B5, B6, B8, B9, B11, and FIG. Reaction group: A5, A6, A7, A9, A10 of Figure 4) were selected, proteins were extracted and purified and separated on gel with a 12.5% concentration gradient. The isolated protein was transferred to nitrocellulose membrane in a solution containing 50 mM Tris, 130 mM glycine, 0.05% SDS, 12.5% methanol. TBST (Tris buffered saline tween 20) in which 5% skim milk powder was dissolved was added to the transferred membrane to block nonspecific binding, and primary antibodies anti-p90RSK and anti-phospho p90RSK were diluted 1: 1500 in TBST for 2 hours. Reacted with the membrane. Subsequently, the antibody was reacted with a secondary antibody anti-rabbit IgG-HRP diluted 1: 5000 for 1 hour, and again subjected to ECL (Enhanced chemiluminance), which was then exposed to an X-ray film in the dark, and the results are shown in FIG. 4.

As shown in Figure 4, when the expression ratio of the total p90RSK and phospho-p90RSK was measured, when the expression rate of phospho-p90RSK is high (2.7 / 2.9) is low when the expression rate of phospho-p90RSK (0.6 / 1.7) Compared to that, the anticancer drug resistance was low, and the results of the clinical sample showed that p90RSK could be used as a biomarker for anticancer drug resistance.

1A shows breast cancer cell lines (MCF7, MCF7-s, MDA-MB-231, MDA-MB-231s, MDA-MB-436, MDA-MB-468, ZR-75-1, BT474, HS578T, T47D) and controls Western blot of the protein extracted from MCF-10A (breast cell line),

Figure 1b is the result of the Western blot measured by using an optical densitometer and quantified,

FIG. 1C is a logarithmic representation of the expression rate of the phosphorylated form (Phospho) compared to the total form based on the numerical value described in FIG. 1B.

2A shows breast cancer cell lines (MCF7, MCF7-s, MDA-MB-231, MDA-MB-231s, MDA-MB-436, MDA-MB-468, ZR-75-1, BT474, HS578T, T47D) and controls Dose rubin treatment to MCF-10A (breast cell line) is a result shown by measuring the sensitivity,

FIG. 2B shows the results of FIG. 2A unified with only one experimental value except for the control part in one graph.

Figure 3a is the result of analyzing the activity level of the mechanism of action of p90RSK using the ingenuity pathway analysis system (A: activation, P: phosphorylation),

Figure 3b is a schematic diagram of the MAPK pathway (phospho-p90rsk) involved in cell growth through Western blot,

4 is a result of analyzing the expression ratio of p90RSK and phospho-p90RSK from the tissue of breast cancer patients in association with sensitivity to anticancer drug resistance.

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Claims (15)

A biomarker composition for diagnosing anticancer drug resistance in a breast cancer patient, comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1, The anticancer agent is anthracycline-based anticancer agent, composition. An antibody that specifically binds to a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an immunogenic fragment thereof and a phosphorylated amino acid sequence of SEQ ID NO: 1 A kit for diagnosing cancer resistance in a breast cancer patient, comprising an antibody that specifically binds to a protein or an immunogenic fragment thereof. The method of claim 3, The anticancer agent is anthracycline-based anticancer agent, kit. The method of claim 3, The resistance diagnostic kit includes a secondary antibody conjugate conjugated to a label that is developed by reaction with a substrate; A color substrate solution to be colored and reacted with the label; Washing liquid; And an enzyme reaction stopper. The method of claim 5, The label of the secondary antibody conjugate is selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase, colloid gold, fluorescein, and dye, Kit. The method of claim 5, The chromogenic substrate is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], and OPD (o-phenylenediamine) The kit of which is selected from the group consisting of. A method of measuring the phosphorylation level of a protein consisting of the amino acid sequence of SEQ ID NO: 1 using an antigen-antibody binding reaction from a sample to provide information necessary for diagnosing anticancer drug resistance in a breast cancer patient. The method of claim 8, The anticancer agent is an anthracycline-based anticancer agent. The method of claim 8, wherein 1) coating the sample and the protein of the control to the fixture; 2) consisting of the amino acid sequence of SEQ ID NO: 1 in the fixture An antibody that specifically binds to a protein or immunogenic fragment thereof and to be phosphorylated with the amino acid sequence of SEQ ID NO: Performing an antigen-antibody binding reaction by adding an antibody that specifically binds to a protein or immunogenic fragment thereof; 3) detecting the antigen-antibody binding reactant produced through the antigen-antibody binding reaction using a secondary antibody conjugate and a color substrate solution; And 4) comparing the detection results for the sample and the control; / RTI &gt; The method of claim 10, The sample of step 1) is tissue isolated from a breast cancer patient. The method of claim 10, Wherein the fixture of step 1) is selected from the group consisting of nitrocellulose membranes, PVDF membranes, well plates synthesized from polyvinyl or polystyrene resins, and glass slide glass. The method of claim 10, The antigen-antibody binding reaction of step 2) is performed by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunoassay. The method is selected from the group consisting of immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation method. The method of claim 10, The marker of the secondary antibody conjugate of step 3) is selected from the group consisting of horseradish peroxidase (HRP), basic dephosphatase, colloidal gold, fluorescent substance, and pigment. The method of claim 10, The color substrate of step 3) is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], and OPD (o -phenylenediamine).

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