JP2009168514A - Breast cancer marker and breast cancer affection recognizing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide highly reliable breast cancer markers found out through a marker search wherein transcriptomic analysis and proteomic analysis reflect each other with a close relationship and methods for recognizing affection on breast cancer. <P>SOLUTION: An oestrogen receptor positive breast cancer marker is provided containing alpha-basic crystallin. A breast cancer marker is provided containing 6-phosphogluconolactonase and/or F-actin capping protein alpha-2 subunit. A method is provided for recognizing affection on breast cancer by using the alpha-basic crystallin as protein for the positive breast cancer marker. Another method is provided for recognizing affection on breast cancer by using the 6-phosphogluconolactonase and/or the alpha-2 subunit as protein for the cancer marker. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to techniques for clinical diagnosis, screening, follow-up, and classification, and specifically to a breast cancer marker and a method for identifying breast cancer morbidity.

  Breast cancer is the most common malignant disease, accounting for 18% of women's cancer (Non-Patent Document 1: Lancet Oncol. 2001, 2, 533-543). For example, in Singapore, three women are diagnosed with breast cancer a day, and the number of breast cancer patients shows a surprising increase of 3.7% every year (Non-Patent Document 2: Chia, KS, Du, WB, Sankaranarayanan, R., Sankila, R. et al., Int. J. Cancer 2004, 108, 761-765). The most effective method of cancer treatment is early detection. Early detection is very advantageous in terms of increasing survival rate and reducing the number of operations and drug treatment (Non-patent Document 3: Nat. Rev. Cancer 2003, 3, 243-252).

  Due to the rapid progress of genome-related technology, efforts have been started for early detection at the molecular level (Non-patent document 4: Curr. Opin. Oncol. 2001, 13, 415-419, Non-patent document 5: Toxicology 2005, 206, 91-109, Non-patent document 6: Br. J. Cancer 2000, 82, 270-277, Non-patent document 7: Nat. Rev. Cancer 2003, 3, 267-275, Non-patent document 8: Arch. Pathol Lab Med. 2002, 126, 1518-1526).

  For example, certain genes such as BRCA1 and BRCA2 have been found to be important as markers for breast cancer. Women with such genes develop breast cancer with a probability of 80% in their lifetime (Non-Patent Document 9: Fam. Cancer 2006, 5, 135-142).

  On the other hand, Non-Patent Document 10: Klemenz, R., Scheier, B., Muller, A., Steiger, R. et al., Verh. Dtsch. Ges. Pathol. 1994, 78, 34-35. Is similar to small heat shock protein (heat shock protein beta-5), and the same companion HSP27 shows upregulation in many breast cancers, but CRAB is often absent It is described.

  In addition, there is a report that CRAB expression may be related to the basal-like subtype (ER- / ERb2-) of human breast cancer (Non-Patent Document 11: Moyano, JV, Evans, JR, Chen, F., Lu, M. et al., J Clin. Invest 2006, 116, 261-270., Non-Patent Document 12: Gruvberger-Saal, SK, Parsons, R., J Clin. Invest 2006, 116, 30- 32.).

  Furthermore, Non-Patent Document 13: Shen, D., Chang, HR, Chen, Z., He, J., Lonsberry, V., Elshimali, Y., Chia, D., Seligson, D., Goodglick, L. NX, Nelson, SF, Gornbein, JA, Biochem. Biophys. Res. Commun. 2005, 326, 218-227.

  In recent years, because of the classification system based on molecular data, mRNA microarrays were used to discover new breast cancer subtypes that were overlooked by conventional methods, and a genome database of sporadic breast cancer and normal tissues from Asian patients was created. (Non-Patent Document 14: Cancer Res. 2004, 64, 2962-2968, Non-Patent Document 15: Clin. Cancer Res. 2004, 10, 5508-5517).

  In parallel to genome research, proteome research has also comprehensively profiled proteins expressed in cells at one time (Non-patent Document 16: Biotechnology (NY) 1996, 14, 61-65, non-patent literature). Patent Document 17: Nature 2003, 422, 198-207).

  Today, genomics and proteomics have become very important tools for cancer biomarker discovery.

Parkin, D. M., The Lancet Oncology. 2001, 2, 533-543. Chia, K.S., Du, W.B., Sankaranarayanan, R., Sankila, R. et al., International Journal of Cancer 2004, 108, 761-765 Etzioni, R., Urban, N., Ramsey, S., McIntosh, M. et al., Nature Reviews Cancer 2003, 3, 243-252. Gerrero, M. R., Weber, B. L., Current Opinion in Oncology 2001, 13, 415-419. Pole, J. C., Gold, L. I., Orton, T., Huby, R. et al., Toxicology 2005, 206, 91-109. Daidone, M. G., Luisi, A., Martelli, G., Benini, E. et al., British Journal of Cancer 2000, 82, 270-277. Wulfkuhle, J. D., Liotta, L. A., Petricoin, E. F., Nature Reviews Cancer 2003, 3, 267-275. Rai, A. J., Zhang, Z., Rosenzweig, J., Shih, I. et al., Archives of Pathology and Laboratory Medicine 2002, 126, 1518-1526. Foulkes, W.D., Familial Cancer 2006, 5, 135-142. Klemenz, R., Scheier, B., Muller, A., Steiger, R. et al., Verhandlungen der Deutschen Gesellschaft fur Pathologie 1994, 78, 34-35. Moyano, J. V., Evans, J. R., Chen, F., Lu, M. et al., The Journal of Clinical Investigation 2006, 116, 261-270. Gruvberger-Saal, S. K., Parsons, R., The Journal of Clinical Investigation 2006, 116, 30-32. Shen, D., Chang, HR, Chen, Z., He, J., Lonsberry, V., Elshimali, Y., Chia, D., Seligson, D., Goodglick, L., Nelson, SF, Gornbein, JA, Biochemical and Biophysical Research Communications 2005, 326, 218-227. Yu, K., Lee, C. H., Tan, P. H., Hong, G. S. et al., Cancer Research 2004, 64, 2962-2968. Yu, K., Lee, C. H., Tan, P. H., Tan, P., Clin. Cancer Research 2004, 10, 5508-5517. Wilkins, M.R., Pasquali, C., Appel, R.D., Ou, K. et al., Biotechnology (N.Y.) 1996, 14, 61-65. Aebersold, R., Mann, M., Nature 2003, 422, 198-207.

  Non-Patent Document 10 describes that CRAB is often not present there (ie, cancerous part). However, the test of the statistical significance about the expression level of CRAB in cancer patient-derived tissue and healthy subject-derived tissue has not been performed. Therefore, this document does not suggest that CRAB is useful as a breast cancer marker.

  Non-Patent Document 11 reports that CRAB expression may be related to the basal-like subtype (ER- / ERb2-) of human breast cancer. The basal-like subtype belongs to a classification that does not feature estrogen positivity. Moreover, the expression level of CRAB in cancer patient-derived tissue and healthy subject-derived tissue is not described.

Cancer biomarker studies usually face two dilemmas.
The first is the selection of experimental samples. It is known to be limited by the use of cell lines or tissue samples. The advantage of using a cell line is that a sufficient amount of the same sample can be easily obtained, however, when isolated from the natural tissue microenvironment, primary cells change their various properties over time. To do. For this reason, cell line studies are difficult to interpret. On the other hand, tumor tissue contains stroma, fibroblasts, immune bodies, blood vessels, blood, etc. that are present in varying amounts depending on the tissue, which significantly hinders proteome analysis.

  The second is the choice of platform technology. Compared to proteomics, gene microarray analysis is clearly superior in sensitivity and throughput and is outstanding in the profile of many primary tissue samples. Nevertheless, the final target of a biomarker is a protein. However, genetic analysis results do not always reflect the situation at the protein level.

  An object of the present invention is to provide a highly reliable breast cancer marker found by marker search in which transcriptomic analysis and proteomic analysis are reflected in a close relationship with each other. Another object of the present invention is to provide a method capable of identifying the morbidity of breast cancer using such a breast cancer marker.

  To solve the above problems, the inventors have developed an integrated proteomic / transcriptomic method using both human cell lines and tissue samples. By this method, we identified nine breast cancer biomarker candidates. Furthermore, cross validation from the analysis of immunohistochemical staining using 2-DE / MS analysis and tissue microarray was performed, and among the four proteins that passed the validation test, CRAB, 6PGL, and CAZ2 were newly developed. Determined as a breast cancer marker.

The present invention includes the following inventions.
The following (1) is directed to a protein of a breast cancer marker that shows down-regulation in estrogen receptor positive breast cancer.
(1) An estrogen receptor-positive breast cancer marker including Alpha basic-crystallin (CRAB).

The following (2) is directed to a breast cancer marker protein that shows up-regulation in breast cancer.
(2) A breast cancer marker containing 6-phosphogluconolactonase (6PGL) and / or F-actin capping protein alpha-2 subunit (CAZ2).

  The following (3) to (6) relate to a method for identifying the incidence of breast cancer. In the present invention, morbidity broadly refers to a morbid state, and identifying morbidity of breast cancer is used in the sense including performing breast cancer detection, diagnosis, monitoring, staging, and prognosis determination.

The following (3) and (4) relate to a method for identifying the incidence of breast cancer using the breast cancer marker described in (1) above.
(3) Alpha Basic—A method for identifying breast cancer morbidity by using crystallin as an estrogen receptor positive breast cancer marker protein.
(4) measuring the level of the protein in a sample derived from an individual who is to be identified as having breast cancer;
Comparing the measured level obtained with the normal level of the protein,
One of the indicators that the subject individual is highly likely to have estrogen receptor-positive breast cancer is that the obtained measurement level is lower than the normal level (3 ) For identifying the incidence of breast cancer.

  The normal level of the protein may be the level of the protein in a sample serving as a control for a cancerous sample, and may be a level in a normal sample derived from a healthy individual, or a cancer derived from a patient suffering from breast cancer. It may be a level in a normal sample that is not sex.

The following (5) and (6) relate to a method for identifying the incidence of breast cancer using the breast cancer marker described in (2) above.
(5) A method for identifying the incidence of breast cancer by using 6-phosphogluconolactonase and / or F-actin capping protein alpha-2 subunit as a protein of a breast cancer marker.
(6) measuring the level of the protein in a sample derived from an individual who is to be identified as having breast cancer;
Comparing the measured level obtained with the normal level of the protein,
The obtained measured value level is higher than the normal level as one of the indicators that the subject individual is likely to have breast cancer, according to (5). A method of identifying the incidence of breast cancer.

  The normal level of the protein may be the level of the protein in a sample serving as a control for a cancerous sample, and may be a level in a normal sample derived from a healthy individual, or a cancer derived from a patient suffering from breast cancer. It may be a level in a normal sample that is not sex.

The method for identifying the incidence of breast cancer as described above, wherein the sample is breast tissue and serum.
A method for identifying the prevalence of breast cancer, wherein the protein level is measured by a test based on biospecific affinity.

The present invention is also directed to a pharmaceutical composition for the treatment of cancer described in (7) and (8) below. The treatment of cancer includes killing cancer cells and suppressing the growth of cancer cells.
(7) A pharmaceutical composition for inducing a reaction that promotes death of breast cancer cells and / or suppression of breast cancer cell growth by supplying to breast cancer cells, comprising (1) and (2) A pharmaceutical composition comprising at least one antibody that immunospecifically binds to a breast cancer marker selected from the group.

(8) A pharmaceutical composition for promoting an immune response by supplying an immunostimulatory amount to breast cancer cells, comprising a breast cancer marker selected from the group consisting of (1) and (2), Pharmaceutical composition.

  The pharmaceutical compositions of (7) and (8) above can be identified as potential therapeutic agents used in the treatment of breast cancer. Or the pharmaceutical composition of said (7) and (8) can be used as a therapeutic agent used for the treatment of breast cancer.

  According to the present invention, it is possible to provide a highly reliable breast cancer marker found by marker search in which transcriptomic analysis and proteomic analysis are reflected in a close relationship with each other. The present invention can also provide a method by which such breast cancer markers can be used to identify morbidity for breast cancer.

<Genomics / proteomics integration technology for cancer marker selection>
The breast cancer marker protein of the present invention has been found by platform technology for selection of cancer markers using genomic and proteomic integration techniques for both cell lines and primary tissue samples.

This technology:
S1: Using the cultured cells of normal and breast cancer cell lines, obtaining information Pc (p) on proteins with different expression levels between normal cell lines and breast cancer cell lines;
S2: converting the protein information Pc (p) into corresponding gene information Gc (p);
S3: obtaining information Gc (g) of a gene whose expression level is different between a normal cell line and a breast cancer cell line from the same cultured cell as the cultured cell;
S4: comparing the gene information Gc (p) with the gene information Gc (g) and selecting a protein exhibiting the same expression tendency between a normal cell line and a breast cancer cell line;
S5: obtaining information Gt of genes whose expression levels differ between normal tissue and breast cancer tissue from normal breast tissue and breast cancer tissue;
S6: The gene information of the selected protein showing the same expression tendency between the normal cell line and the breast cancer cell line is compared with the information Gt of the gene, and the normal cell line / normal tissue and the breast cancer cell line / breast cancer are compared. Further selecting a protein exhibiting the same expression tendency with the tissue.
In addition to these steps, the protein selected as described above can be narrowed down based on the immunochemical reaction.
A series of flow of this tumor marker search method is shown in FIG.

(Proteomics)
In proteomics, a series of proteomic analyzes (S1) by 2DE / MS (ie, a method in which proteins are separated by two-dimensional electrophoresis and the separated proteins are identified by mass spectrometry) are used to identify proteins with different expression levels. Profiling is done. Thereafter, mRNA search corresponding to the proteins having different expression levels is performed (S2).

(S1: Protein profiling)
(Breast cancer / Normal cell line)
A breast cancer cell line and a normal cell line as a control are prepared. Examples of breast cancer cell lines include those that are estrogen receptor positive (eg, MCF-7) and those that are estrogen receptor negative (eg, HCC-38). An example of a normal control cell line is CCD-1059Sk. These cell lines are cultured to prepare cell lysate.

(Protein separation and protein spot quantification)
A portion of the cultured cell line cell lysate is subjected to two-dimensional electrophoresis (2DE). The gel image of the separated protein spots is digitized, and proteins showing different expression between the breast cancer cell line and the normal cell line are examined. For gel image analysis, image analysis software such as PDQuest (Bio-Rad) is used, and spots can be quantified using this software.

(Identification of protein)
Cut out protein spots that show different expression between breast cancer cell lines and normal cell lines. The excised protein spot can be washed, trypsin digested, blotted to an MS sample plate, and identified using a mass spectrometer. In this way, cell line-derived protein information Pc (p) is obtained by proteomics.

(S2: Search for mRNA corresponding to protein information)
From the information Pc (p) relating to proteins whose expression differs between the breast cancer cell line and the normal cell line obtained as described above, mRNAs corresponding to the proteins are searched. In this way, gene information Gc (p) derived from the cell line by proteomics is obtained.

(Genomics)
Also, post-transcriptional regulatory mechanisms in cell development and perturbation are critical events, but protein expression is regulated at the mRNA level, so most proteins are regulated in transcription. In genomics, mRNA profiling with different expression levels from cell lines (S3) and mRNA profiling with different expression levels from tissues (S5) are performed.

(S3: mRNA profiling directly from the cell line)
So, extract the total mRNA from the same batch as the cell lysate prepared as described above,
Profiling of mRNAs with different expression can be performed. mRNA profiling can be performed using a microarray chip. In this way, gene information Gc (g) derived from the cell line by genomics is obtained.

(S5: mRNA profiling from tissues)
On the other hand, total mRNA can be extracted from breast cancer tissue and normal breast tissue (control), and mRNAs with different expression can be profiled. mRNA profiling can be performed using a microarray chip. In this way, gene information Gt derived from the tissue is obtained.

(Genomics / proteomics integrated analysis)
In the genomics / proteomics integrated analysis, the gene information narrowed down by the proteomics is narrowed down using the gene information obtained by genomics.

(S4: Comparison of gene information corresponding to protein profile from cell line and mRNA profile obtained directly from cell line)
The gene information Gc (p) narrowed down by the above proteomics (S1 and S2) and the gene information Gc (g) obtained by genomics (S3) from the cell line are compared, and those that show the same expression tendency are selected. To do. Specifically, among mRNA Gc (p) obtained by 2DE-MS analysis in proteomics and showing different expression between normal cell lines and breast cancer cell lines, normal and obtained from microarray analysis in genomics. Those having the same expression tendency as mRNA Gc (g) showing different expression among breast cancers are extracted, and a protein corresponding to the mRNA showing the same expression tendency is temporarily set as a marker candidate. The candidates extracted in this way are significant candidates in terms of both mRNA level and protein level. The genomics / proteomics integrated analysis has the advantage that the reliability of the found protein as a cancer marker is very high because only gene products having high potential as cancer markers can be sub-selected.

  Specifically, in the narrowing-down method, the consistency of expression tendency between the expression level of the gene corresponding to the protein and the mRNA level obtained directly from the cell line is examined. As an analysis system, it is preferable to use Gene Set Enrichment Analysis (GSEA). This method determines whether a speculatively defined group of genes shows a statistically significant and consistent difference between two biological states (eg, cancer and normal). Thereby, based on mRNA directly obtained from the cell line, mRNA showing an inconsistent expression tendency among mRNA determined from the protein level can be excluded from candidates.

The gene information Gc (g) profile obtained by cell line genomics is filtered to extract genes that show an effective value of a certain amount or more. Then, a list of genetic information Gc (g) is created. This list ranks the differences in gene expression levels between normal cell lines and breast cancer cell lines in order of size.
On the other hand, the spot of protein Pc (p), selected by 2DE gel image comparison and having a significant difference between normal cell line and breast cancer cell line, corresponds to the corresponding mRNA Gc using Affimetrix U133 plus chip annotation and SwissProtIDs. (p) can be mapped and used as a test group for the above list of genetic information Gc (g).

  The agreement between the expression tendency of the gene Gc (g) and the protein Gc (p) means that the expression levels of the gene Gc (g) and the protein Gc (p) with respect to the normal cell line (control) are the same. That is, the expression levels of the gene Gc (g) and the protein Gc (p) are both increased or decreased to the same extent relative to the normal cell line (control).

(S6: Comparison between the profile from the cell line and the profile from the organization)
Candidates that exhibit the same expression tendency at the gene level and the protein level extracted in S4 can be further subjected to cross-validation.
In this cross-validation evaluation, the genetic information Gt derived from the tissue obtained in S5 above is divided into a training mRNA data set and a test mRNA data set, and a normal sample in the training data set is obtained by supervised learning analysis. The test mRNA data set is then differentiated between normal and cancer samples under the same conditions. This test condition allows us to assess the actual performance of the data that is not available for the training mRNA dataset. Specifically, a Support Vector Machine algorithm or the like can be used. In addition, after analysis with the training data set, an independent data set such as a test mRNA data set can be subjected to principal component analysis to check whether accurate separation is possible.

(S7: Immunochemical validation)
The candidate protein that has passed the cross-validation as described above can be further determined from the immunochemical viewpoint and / or other viewpoints to determine a marker protein with higher reliability. These validations may be performed by 2DE / MS analysis, Western blotting analysis, TMA (tissue microarray) analysis, or the like. Validation from an immunohistochemical viewpoint, such as TMA analysis, is very preferable in that it can assure the reliability of breast cancer biomarkers to the maximum.

<Tumor marker for breast cancer>
As a tumor marker for breast cancer, the present invention provides three proteins: alpha basic-crystallin (CRAB), 6-phosphogluconolactonase (6PGL), and F-actin capping protein alpha. -2 subunit (F-actin capping protein alpha-2 subunit: CAZ2) is provided. The breast cancer tumor marker of the present invention is a very reliable tumor marker found by the above-described genomics / proteomics integration technique and various validations.

  In the tumor marker of the present invention, the significant difference between the breast cancer cell line and the normal cell line is p <0.001 in the t-test. Usually, a significant difference is observed when the p-value in the t-test is less than 0.05, more preferably less than 0.01. Therefore, the marker of the present invention is a highly reliable marker in that a further significant difference is recognized between the breast cancer cell line and the normal cell line.

  Among the proteins provided by the present invention as a tumor marker, CRAB is a protein that shows down-regulation in breast cancer. In particular, since it specifically shows down-regulation in estrogen receptor positive (ER +) breast cancer tissue, it is useful as a breast cancer marker for estrogen receptor positive (ER +) breast cancer.

  Among the proteins provided by the present invention as tumor markers, 6PGL and CAZ2 are proteins that show up-regulation in breast cancer.

  These proteins can be isolated and purified by any protein purification technique selected by one skilled in the art. For example, techniques including chromatography such as ion exchange, affinity, and size exclusion column chromatography, centrifugation, differential solubility, and electrophoresis can be used.

<Method for identifying the incidence of breast cancer>
The present invention also provides a method for identifying breast cancer disease by using at least one protein selected from the above proteins as a tumor marker for breast cancer.

  In the method of the present invention, a sample derived from an individual who is a subject to be identified as having breast cancer is prepared, and the level of at least one protein selected from the proteins is measured in the sample. The obtained measurement level is compared with the normal level. The normal level is the protein level in a sample serving as a control for a cancerous sample of at least one protein selected from the above proteins. Here, the sample serving as a control for the cancerous sample is not particularly limited as long as it is a non-cancerous sample, and may be a sample derived from a healthy individual or a normal sample derived from a patient suffering from breast cancer.

  In this invention, it does not specifically limit as a sample originating in the individual used as the object which should identify the affliction of breast cancer. Examples include cells, tissues, body fluids, and tissue extracts. In particular, among these samples, a sample derived from breast tissue is preferable. Cells and tissues include tissue biopsy materials and autopsy materials. The body fluid includes blood, body secretions and the like. The blood includes whole blood, plasma, serum and the like. Tissue extract refers to tissue homogenated or solubilized by methods known to those skilled in the art. Among these exemplified samples, it is preferable to use breast tissue as a sample.

  Tumor marker measurements measured in a sample containing a subject's individual cells, tissues, body fluids, and tissue extracts, preferably non-cancerous samples containing the same type of cells, tissues, body fluids, and tissue extracts Compared to the level in That is, if the measured tumor marker is of the subject's individual breast tissue, this level is preferably compared to the level of normal breast tissue that is not cancerous.

  If the protein is CRAB and the obtained measurement level is lower than the normal level, the individual to whom breast cancer is to be identified is likely to have cancer. It can be one of the indicators. CRAB is particularly useful in identifying the morbidity of estrogen receptor positive (ER +) breast cancer because it specifically down-regulates in estrogen receptor positive (ER +) breast cancer tissue.

  As a result of quantitative analysis of the S1 step, this protein showed 1/26 times (molar basis) expression level in the estrogen receptor-positive breast cancer cell line than in the normal cell line. Therefore, the degree of decrease in the measured value level may be determined based on the degree to which the measured value level is 1/10 or less, preferably 1/15 or less (molar basis) of the normal value level.

  When the protein is 6PGL or CAZ2, it is possible that the individual to whom breast cancer is to be identified is suffering from cancer that the obtained measurement level is higher than the normal level It can be one of the indicators of high nature.

  Of these proteins, as a result of quantitative analysis in the S1 step, 6PGL shows an expression level 48 times (molar basis) greater in the breast cancer cell line than in the normal cell line, and CAZ2 is in the normal cell line in the breast cancer cell line. The expression level was 23 times or more (on a molar basis) greater than that in the line. Therefore, the degree of increase in the measured value level is such that the measured value level is 15 times or more, preferably 20 times or more (molar basis) of the normal level in the case of 6PGL, and the measured value level in the case of CAZ2. The standard is a level that is at least 10 times the normal level, preferably at least 15 times (on a molar basis).

  The protein level is preferably measured by a test based on biospecific affinity. The test based on biospecific affinity is a method well known to those skilled in the art and is not particularly limited, but immunoassay and immunostaining are preferred. Specifically, Western blot, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation method, precipitation reaction, in-gel diffusion precipitation reaction, immunodiffusion method, aggregation measurement, complement binding assay, immunoradiometric assay, fluorescent immunoassay, Immunoassays including competitive and non-competitive assay systems such as protein A immunoassays; immunostaining including tissue or cell staining, immunoelectron microscopy, tissue microarray (TMA). In these methods, the presence of an antibody that binds to a tumor marker in an individual's sample is detected. Specifically, it is carried out by contacting a sample with the antibody under conditions capable of forming an immune complex consisting of a tumor marker protein to be measured and an antibody of the protein in an assay medium or on a tissue section. Is called. More specific protocols can be easily determined by those skilled in the art.

  The level of the protein is preferably measured by a test based on biospecific affinity as described above, but may be measured by other protein quantification methods. For example, the isotope labeling method is an excellent method for quantitativeness. In this case, using a sample prepared with a known level of the above protein or an appropriate sample such as a normal sample as a control sample, the measurement is performed by examining the difference in the abundance of the protein from the sample of the subject individual. It can be carried out.

  In the method of the present invention, the tumor marker of the present invention may be measured alone or in combination with any other tumor marker. Accordingly, the methods of the present invention may include measuring the levels of other tumor markers as well as the levels of the tumor markers of the present invention.

  The tumor marker of the present invention may be used for the purpose of cancer detection, diagnosis, monitoring, staging and prognosis determination. Preferably, it may be used for grasping the dynamics of the tumor. For example, when chemotherapy or radiation therapy is performed on a tumor, it may be used to determine how effective the treatment is. Moreover, when excision surgery is performed on a tumor having a high tumor marker value, it may be used for post-operative follow-up.

<Pharmaceutical composition>
The present invention also includes among the tumor markers of the present invention, a protein having a high expression level (ie, a protein selected from 6PGL and CAZ2) and a protein having a low expression level (ie, CRAB) in breast cancer patients. It is directed to a pharmaceutical composition for treating breast cancer by using selected tumor markers.

  One form of the pharmaceutical composition is a pharmaceutical composition for inducing a response that promotes death of cancer cells and / or suppression of growth of cancer cells by supplying the cancer composition to the cancer cells. A pharmaceutical composition comprising at least one antibody that immunospecifically binds to a marker. Antibodies include polyclonal antibodies, monoclonal antibodies, and antibodies prepared by molecular biological techniques. Here, the antibody may be any substance that binds widely immunospecifically, and an antibody fragment or an antibody fusion protein may also be used. In either case, antibody preparation is carried out by methods well known to those skilled in the art.

  Another form of the pharmaceutical composition is a pharmaceutical composition for promoting an immune response by supplying an immune stimulating amount to a cancer cell, the pharmaceutical composition comprising the tumor marker of the present invention. It is. Here, the immunostimulatory amount refers to the amount of an antigen capable of eliciting a desired immune response for the treatment of cancer, and is determined by a method well known by those skilled in the art. According to this form, the cancer is treated using a method well known to those skilled in the art, known as so-called cancer vaccine therapy.

  The pharmaceutical composition contains the antibody or tumor marker as an active ingredient, and may further contain a pharmaceutically acceptable diluent, carrier, excipient, and the like. The pharmaceutical composition can be identified as a potential therapeutic agent used in the treatment of cancer. Alternatively, the pharmaceutical composition can be applied as a therapeutic agent used in the treatment of cancer.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Cell line and primary organization]
The cell lines and primary structures used in the present invention are as follows.
(Cell line)
Breast cancer cell line:
ER positive human breast cancer cell line MCF-7 (ATCC: HTB-22)
ER-negative human breast cancer cell line HCC-38 (ATCC: CRL-2314)
Normal cell line:
CCD-1059Sk (ATCC: CRL-2072)
(Primary organization)
Breast cancer tissue:
Obtained from Singapore National Cancer Center Tissue Repository Normal breast tissue:
Obtained from cosmetic surgery patients

[Protein expression profiling from cell lines]
First, cell line proteome analysis by 2DE / MS was used. Separate all cell lysates from breast cancer cell lines (MCF-7 and HCC-38) and normal cell lines (CCD-1059Sk) as controls using pH 4-7 or pH 6-9 narrow range IPG strips, and And subjected to two-dimensional electrophoresis (2DE) using 10 w / w% isocratic SDS-PAGE slab gel. In order to minimize variability between gels, electrophoresis was performed simultaneously using a Dodeca cell (Bio-Rad).

  Protein spots were fluorescently stained using Deep Purple (GE HealthCare), and quantitative gel image analysis was performed using PDQuest 7.3 image analysis software (Bio-Rad). Furthermore, 2,548 protein spots were cut out from the 2D gel using the Xcise (Shimadzu Biotech) system.

  The excised protein spots were trypsin digested, MALDI-TOF MS measurement (positive ion / reflectron mode) was performed with AXIMA-CFR plus mass spectrometer (Shimadzu Biotech), and 1,724 protein spots (484 Corresponding to different protein species).

Profiling of proteins with different expression was performed.
In order to compare the protein profiles of MCF-7 and CCD-1059Sk, Student's t-test on the staining intensity of protein spots was performed. Identified 200 protein spots from both pH ranges (108 spots at p <0.0035 (pH 4-7 gel) and 82 spots at p <0.0014 (pH 6-9 gel)) and finally In MCF-7, 78 proteins expressed more and 32 proteins expressed less than CCD-1059Sk were derived.

  In order to compare the protein profiles of HCC-38 and CCD-1059Sk, Student's t-test was performed in the same manner. Then, the top 200 protein spots showing different expression were identified (p <0.0067 (pH 4-7 gel) and p <0.00094 (pH 6-9 gel)), and finally the CCD spot in CCD-1059Sk in HCC-38. In comparison, 81 proteins that were highly expressed and 31 proteins that were expressed less were derived.

[Gene expression profiling from cell lines and tissues]
In parallel with the same batches of normal and breast cancer cell lines prepared by protein expression profiling from cell lines, mRNA profiling shown in S3 of FIG. 1 was performed using a microarray chip U133_plus GeneChip (Affymetrix). As a result, approximately 35,000 mRNAs were detected simultaneously from each chip.
Similarly, mRNAs showing different expression were also profiled for breast cancer tissue and normal breast tissue using a microarray chip U133_plus GeneChip (Affymetrix).

  Specifically, total RNA was extracted from cell lines and tissues using Trizol reagent (Invitrogen) and hybridized to a microarray chip U133_plus GeneChip (Affymetrix). The measured original data files were stored in a central database and quality controlled using GeneData Refiner software (GeneData). The refined profile was normalized to an intensity value of 500. The gene expression data of the primary tissue of this cancer can be obtained from the gene expression bank (GEO) accession number GSE2294.

[Statistical integration of proteomics and gene expression]
Protein IDs identified by protein expression profiling from the cell line were matched to the corresponding mRNA microarray (U133_plus) probe using SwissProt ID (www.affimetrix.com).
Normal cells obtained by gene expression profiling from cell lines and tissues from mRNA information corresponding to proteins showing different expression between normal cell lines and breast cancer cell lines obtained by protein expression profiling from cell lines Lines and breast cancer cell lines or mRNAs that differed in expression between normal breast and breast cancer tissues were extracted.

About 78 proteins that express more and less than 32 proteins expressed in MCF-7 than CCD-1059Sk:
Using Affmetrix's U133_plus chip annotation and Swiss Prot ID, these proteins are mapped to their corresponding mRNAs and two sets of microarray probes (ie, corresponding to proteins that are more highly expressed in MCF-7 as the first set) A set of 198 probes (MCF-7 up-regulated) and a second set of 84 probes (MCF-7 down-regulated) corresponding to proteins less expressed in MCF-7 were identified. .

All the probes (˜50K) obtained by S3 in FIG. 1 were ranked according to the strength of the difference in gene expression level between MCF-7 and CCD-1059Sk (ie, arranged in order).
Then, using gene assembly enrichment analysis (GSEA), each of the two MCF-7 up-regulated probe sets and the MCF-7 down-regulated probe set is present at any position in the ranked list. (Ie distribution) was examined. The results are shown in FIG. 2 (A) (UP_MCF7: MCF-7 up-regulated probe set, Down_MFC7: MCF-7 down-regulated probe set, Enrichment Score: enrichment score).

  As shown in FIG. 2 (A), the MCF-7 up-regulated probe set was significantly enriched in the gene region (p <0.001) that showed an increased expression level in MCF-7 cells compared to CCD-1059Sk. It can be seen that the same expression tendency was shown between the proteomic data and the genomic data. Similarly, the MCF-7 down-regulated probe set was also significantly enriched in the gene region (p <0.001) that showed decreased expression levels in MCF-7 cells compared to CCD-1059Sk.

About 81 proteins that are highly expressed in HCC-38 and 31 proteins that are expressed less than CCD-1059Sk:
As above, a set of 195 probes corresponding to proteins more expressed in HCC-38 (HCC38 up-regulated) and a set of 71 probes corresponding to proteins less expressed in HCC-38 (HCC38 down- GSEA was conducted. The results are shown in FIG. 2 (B) (UP_HCC38: HCC38 up-regulated probe set, Down_HCC38: HCC38 down-regulated probe set, Enrichment Score: enrichment score).

  As shown in FIG. 2 (B), the HCC38 up-regulated probe set was significantly enriched in the gene region (p <0.001) that showed an increased expression level in HCC38 cells compared to CCD-1059Sk. The HCC38 down-regulated probe set was also significantly enriched in the gene region (p <0.001) that showed a decreased expression level in HCC38 cells compared to CCD-1059Sk.

  These results indicate that the majority of the proteins with different expression levels (79-82%) identified in the 2DE / MS analysis are also associated with accompanying changes in mRNA expression levels. In order to further narrow down the proteins showing consistent expression trends, 87 proteins selected in comparison of expression levels in MCF-7 and CCD-1059Sk and selection in comparison of expression levels in HCC-38 and CCD-1059Sk Each of the 92 proteins was listed, and they were combined into one list containing 30 proteins that are less expressed in cancer cell lines and 105 proteins that are more expressed in cancer cell lines.

Next, the biomarker candidates were prioritized by comparing the genes corresponding to the proteins thus narrowed down to genes that showed differential expression between breast cancer tissue and normal breast tissue samples at the gene expression level. .
39 primary tissue samples (normal samples: 7, cancer samples: 32) were constructed with training mRNA datasets, using the Support Vector Machine (SVM) machine learning algorithm in combination with 100 cross-validations, The following nine biomarkers were identified that can be reliably identified.

Annexin I (ANX1_HUMAN)
Alpha basic-crystallin (CRAB_HUMAN)
6-phosphogluconolactonase (6PGL_HUMAN)
F-actin capping protein alpha-2 subunit (CAZ2_HUMAN)
Type II cytoskeletal 7 (K2C7_HUMAN)
Lamin B1 (LAM1_HUMAN)
Transketolase (TKT_HUMAN)
Serine / threonine-protein kinase PAK 2 (PAK2_HUMAN)
26S proteasome non-ATPase regulatory subunit 2 (PSD2_HUMAN)
Of these, ANX1 and CRAB showed decreased expression in breast cancer cell lines and primary tissues, and the other seven proteins showed increased expression.

  Furthermore, in order to validate these 9 types of breast cancer marker candidates, by performing principal component analysis (PCA) of mRNA expression of 9 types of breast cancer marker candidates, 36 tissue samples (normal samples: 5, cancer samples: 31). It was evaluated whether the test set can be further classified. The results are shown in FIG. 3 (Comp. 1: first principal component axis, Comp. 2: second principal component axis, Comp. 3: third principal component axis, Normal: normal sample, Tumor: cancer sample). As shown in FIG. 3, nine normal breast cancer marker candidates clearly distinguished normal samples from cancer samples.

  In this way, nine breast cancer marker candidates were identified. Furthermore, verification by validation from tissue microarray analysis was performed. As a result, among the nine candidates, four candidates ANX1, CRAB, 6PGL, and CAZ2 passed the validation test. Nearly half of breast cancer marker candidates have been successfully validated, indicating how systematic integration of proteomics and transcriptomics from cell lines and primary tissue samples is advantageous in the discovery of cancer biomarkers Indicates.

[Analysis by immunohistochemical staining using tissue microarray]
Hereinafter, validation using tissue microarray analysis (TMA) will be described in detail. TMA analysis was performed on the above nine proteins (commercially available antibodies were used for ANX1, CRAB, K2C7, LAM1 and PAK2, and customized antibodies were used for 6PGL, CAZ2, TKT and PSD2). . In the following, among the above four proteins (ANX1, CRAB, 6PGL and CAZ2) finally narrowed down by tissue microarray analysis, the three breast cancer marker proteins of the present invention, CRAB, 6PGL and CAZ2, are mainly used. Show about.
This protocol can be used as one embodiment of a method for identifying the incidence of breast cancer using the breast cancer marker of the present invention.

  Breast cancer tissue microarray slides subjected to tissue microarray analysis each contain 100 arrays of 98 tumor tissues and 2 breast normal tissues (treated with cancer tissues in controls). On the other hand, slides for optimizing staining conditions (for positive control) each contain 100 breast normal tissues or normal cell lines (made with paraffin-embedded blocks).

(CRAB antibody (commercially available))
In order to optimize the conditions of IHC (Immunohistochemical) staining for antibodies, IHC staining optimization was performed on normal breast tissue microarrays. Four different antigen retrievals (to the positive control tissue for CRAB) were performed at different primary antibody concentrations (see Table 1 below). Polyclonal antibodies were tested at 1:50, 1: 100, 1: 200, and 1: 400 dilutions, and monoclonal antibodies were tested at 1: 100, 1: 200, and 1: 400 dilutions. (See Table 1 below). Stained positive control tissue slides were examined by a pathologist. Optimal conditions including specific antibody testing methods and specific primary antibody dilutions were determined after consulting with the pathologist and applied to the breast cancer tissue microarray.

(For 6PGL and CAZ2 antibodies (customized))
The same staining conditions as those of the commercially available antibody are searched. In this case, a paraffin-embedded cell block was used as a positive control.
To create a cell block, MCF7 breast cancer cells were cultured in 75 mL flasks. The cells were trypsinized and spun down to a cell pellet thicker than 5 mm. The cell pellet was immediately incubated with formalin for 20 minutes and embedded in paraffin.
Intracellular localization of the antigen was determined by Western blotting of the fractionated protein extract.

(IHC staining protocol)
solution:
1. Citrate buffer pH6.0
Citric acid 10.5g
Adjust pH to 6.0 with 2M NaOH (approx. 65 mL) Adjust to 5 L with deionized water
(Add citric acid if pH is high, add NaOH if pH is low)
2. Tris-EDTA buffer pH 9.0
Tris base 12g
EDTA 1 g
Adjust to 500 mL with deionized water
Adjust to pH 9 with 0.2M HCl
3. DAKO buffer pH6.0
60ml Dako (10x) (Cat. S2369) Adjust to 600mL by adding deionized water to target retrieval. DAKO buffer pH9.0
60ml Dako (10x) (Cat. S2367) Adjust to 600mL by adding deionized water to target retrieval. TBS wash buffer pH7.6 (10x)
0.5M Tris 60.6g
9w / w% NaCl 90g
Adjust to 1L by adding deionized water, and adjust to pH 7.6 by adding HCl.

I. Deparaffinization Prior to deparaffinization, the array slides were kept at room temperature for 60 minutes in the horizontal state or oven heated at 60 ° C. for 20 minutes. The slides were arranged in a metal tray and the following steps were performed.
1. The slide was immersed in xylene for 5 minutes and further immersed in fresh xylene for 5 minutes.
2. The slide was immersed in 100% ethanol for 5 minutes.
3. The slide was immersed in 95 v / v% ethanol for 5 minutes.
4). The slide was immersed in 70 v / v% ethanol for 5 minutes.
5. The slide was washed with running water.

II. Antigen retrieval method A (Pressure Cook) Citrate buffer (heated in a high-pressure pot)
1. 2 L of citrate buffer was boiled for 10-15 minutes with a stainless steel pressure cooker.
2. When the buffer boiled, the slide was placed vertically into the pot and the lid was tightened. I waited 3 minutes after the two buttons on the lid popped up. (The slide was inserted so that it did not touch the cooker button directly.)
After 3.3 minutes, the pot was cooled by running water in the sink. The knob was turned to release the pressure. After releasing the pressure, the lid was opened and the slide was cooled with running water.

Method B (Tris-EDTA) Tris-EDTA buffer (heated in microwave)
1. 600 ml of buffer was poured into a plastic jar.
2. Slides were placed in a plastic tray and placed in a buffered jar.
3. The jar was wrapped with plastic film and several holes were drilled at the top.
4). Put the jar in the microwave. The power was set to 5 (power was an intermediate level of 5 out of 10) and the time was set to 25 minutes (the buffer was not clear but clouded).
5. The jar was moved into the sink and allowed to stand under running water. The solution became clear again.
6). After cooling, the slide was transferred into a container with water.

Method C (Dako6 Buffer) DAKO pH6.0 buffer The same procedure as in Method B was performed using this buffer.

Method D (Dako9 Buffer) DAKO pH 9.0 buffer The same procedure as in Method B was performed using this buffer.

III. Immunostaining The slide was dried and surrounded around the tissue to be stained with dacopen (DAKO, S2002). A circle surrounded by a pen around the tissue serves as a barrier to prevent liquids such as antibody solutions and wash buffers from flowing out of the circle.
2. The slide was dried and a block solution (DAKO, S2023) was dropped into the tissue. Incubated for 10 minutes.
Care was taken not to generate bubbles in the solution during the incubation.
3. The solution on the slide was discarded and rinsed with deionized water. The rack was further washed with running water for 5 minutes.
4). Place the slide horizontally in a plastic tray and pour TBS for 5 minutes.
5. The primary antibody (Ab) was diluted with an antibody diluent (DAKO, S0809).
6). The slide was drained with a gentle hit and 200 μL of diluted Ab was added to the tissue and incubated for 30 minutes (to avoid air bubbles).
7). The slide was taken out, dipped in TBS and drained.
8). TBS was poured onto the slide and waited 5 minutes. During this time, TBS was added as appropriate so that the slide did not dry.
9. Drain the water while lightly hitting the slide on the hand towel. A white bottle (Dako REALTM EnVision ™ HRP Rabbit / Mouse) was taken out from the envision kit (DAKO, S5007), and a few drops were dropped on the tissue and allowed to stand for 30 minutes.
10. The slide was drained and washed with TBS. TBS was poured onto the slide and waited for 5 minutes.
11. 1 ml of substrate buffer was mixed with 20 μL of DAB (3,3′-diaminobenzdine: Toxic). Mixing was done before use. Substrate buffer and DAB are included in the envision kit (DAKO, S5007).
12 The slide was washed with TBS, tapped lightly and drained, and the DAB mixture was dropped onto the slide and incubated for 10 minutes.
13. The slide was washed with d-H2O.
14 The slides were arranged in an H / E rack and allowed to stand for 5 minutes under running water.
15. Slides were placed in haemtoxylin for 2 minutes until the tissue was blue and left in PBS for 10 minutes. The slide was washed with running water for 10 minutes.
16. Slides were dehydrated with a series of solutions: 70 v / v% ethanol, 95 v / v% ethanol, 100 v / v% ethanol x 4, xylene x 2 (each used for 1 minute).
17. The slide was mounted on the mounting machine.
In all steps, the tissue surface remained moist. Otherwise, the dried part will show non-specific staining.

  Table 1 shows the TMA results of breast cancer marker candidates (Candidates) together with the results of 2DE / MS (2DE / MS on Cell lines) in cell lines. A downward arrow indicates that it is down-regulated, and an upward arrow indicates that it is up-regulated. Here, regarding CRAB, Tumor's 2DE / MS on Cell lines showed down-regulation in the estrogen receptor-positive breast cancer cell line, whereas it was up in the estrogen receptor-negative breast cancer cell line. Regulation was shown. Optimal conditions for antibody concentration are shown as dilution ratio of antibody concentration. At the primary antibody suppliers, 6PGL and CAZ2 customized antibodies were prepared at BioGenes. Subcellular location information (discussed below) helps to correlate antibody expression in positive control tissues and tissue microarrays. Cyto represents the cytoplasm, Nucl represents the nucleus, Plasma mem represents the cell membrane, and Mito represents the mitochondria. Information on the subcellular localization of proteins can be obtained from literature on commercially available antibodies (Lit); experiments by the inventors (exp); and predictions using bioinformatics algorithms based on protein sequences. Obtained.

  As for IHC staining for ANX1 in a breast cancer tissue microarray, 88% of tumor tissues showed negative staining. IHC-stained breast tissue (magnification: × 100) is shown in FIG. 5 together with an enlarged view (magnification: × 600) of a part of the tissue (N: normal tissue, T: breast cancer tissue).

  Regarding IHC staining for CRAB in a breast cancer tissue microarray, 96% of tumor tissues showed negative staining (96% of tumor tissues showed negative staining). IHC-stained breast tissue (magnification: x100) is shown in FIG. 6 together with an enlarged view (magnification: x600) of a part of the tissue (N: normal tissue, T: breast cancer tissue). Those showing negative staining in the tumor tissue (94 out of 98 tumor tissues) were all estrogen receptor positive, and showed positive staining in the tumor tissue (4 out of 98 tumor tissues) Were all estrogen receptor negative. On the other hand, all normal tissues of the normal breast tissue microarray showed positive staining. Specifically, it was confirmed that CRAB is strongly expressed in normal duct myoepithelial cells.

  As for IHC staining for 6PGL in the breast cancer tissue microarray, 98% of tumor tissues showed stronger positive staining. IHC-stained breast tissue (magnification: × 100) is shown in FIG. 7 together with an enlarged view (magnification: × 600) of a part of the tissue (N: normal tissue, T: breast cancer tissue). Specifically, 6PGL was widely expressed in the cytoplasm, consistent with bioinformatics predictions. Of these positively stained tumor tissues, 69.3% (61 of 88 tumor tissues) were grade 3 positive staining, 27.3% (24 of 88 tumor tissues) were grade 2 positive staining, 2.3% (88 tumors) Of the tissues, 2) was grade 1 positive staining. However, normal tissues in normal breast tissue microarrays showed grade 1 or grade 2 positive staining, and none showed grade 3 positive staining.

  As for IHC staining for CAZ2 in breast cancer tissue microarray, 69% of tumor tissues showed stronger staining (69% of tumor tissues show stronger positive staining). IHC-stained breast tissue (magnification: × 100) is shown in FIG. 8 together with an enlarged view (magnification: × 600) of a part of the tissue (N: normal tissue, T: breast cancer tissue). Specifically, CAZ2 was mainly expressed in the cytoplasm. Of these positively stained tumor tissues, 15.5% (13 of 84 tumor tissues) were grade 3 positive staining, 16.7% (14 of 84 tumor tissues) were grade 2 positive staining, 36.9% (84 tumors) Of the tissues, 31) had grade 1 positive staining. On the other hand, most of the normal tissues in the normal breast tissue microarray showed negative staining, and in some places, grade 1 positive staining.

[Subcellular localization using Western blotting]
Western blotting was performed on the MCF-7 fractionated protein extract to examine the intracellular localization of the customized antibody. As fractionated protein extracts, cytoplasmic protein extracts, membrane protein extracts, and organelle protein extracts were examined. The result of Western blotting is shown in FIG. In FIG. 4, (i) shows the results for the cytoplasmic protein extract, (ii) for the membrane protein extract, and (iii) for the organelle protein extract.

[Western blotting analysis of tissue samples]
Hereinafter, validation using Western blotting analysis of tissue samples will be described in detail. Western blotting analysis was performed for the above nine proteins (commercially available antibodies were used for ANX1, CRAB, K2C7, LAM1 and PAK2, and customized antibodies were used for 6PGL, CAZ2, TKT and PSD2). ). In the following description, CRAB, 6PGL and CAZ2 are shown among the above four proteins (ANX1, CRAB, 6PGL and CAZ2) finally narrowed down by Western blotting analysis.
This protocol can be used as one embodiment of a method for identifying the incidence of breast cancer using the breast cancer marker of the present invention.

I. Breast tissue samples and protein extracts The breast cancer tissue used in this analysis was obtained from the Singapore National Cancer Center Tissue Repository after examination by two independent histopathologists. Normal breast tissue was obtained from cosmetic surgery patients.
In Western blotting, a mixture of breast cancer tissue extracts from 3 breast cancer patients each having ER + (estrogen receptor) positive and a mixture of breast tissue extracts from 6 normal persons were used.

II. Western blotting Preparation of protein extract The tissue to be analyzed was sliced with a scalpel to obtain an appropriately sized section.
2. Tissue sections are washed 1-2 times in PBS for 30 seconds (the number of washes depends on the degree of contamination. For tissues with large contamination, more washing is required), blood and other contaminants Objects were removed from the tissue surface.
3. Using a pestle and mortar, the tissue sections were ground into finer pieces in liquid nitrogen.
4). Tissue fragments were collected in an Eppendorf tube containing 250 mM aqueous sucrose solution and washed 2-3 times for 30 seconds (the number of washes depends on the degree of contamination with blood).
5. After washing, the mixture was centrifuged at 8000 rpm for 2 minutes, and the supernatant was discarded.
6). After washing with an aqueous sucrose solution, the pellet was taken out and completely pulverized into a fine powder.
7). The powder was collected in an Eppendorf tube and the protein was extracted using the Calbiochem Subcelluar Proteome Extraction Kit (Calbiochem).
8). A large amount of plasma protein was removed from the protein lysate by using Albumin and IgG removal kit (GE Healthcare) followed by Multiple Affinity Removal Column (Agilent Technology).
9. The protein lysate buffer from which a large amount of plasma protein had been removed was replaced with SDS sample buffer (2 w / w% SDS, 20 w / w% Glycerol, 120 mM Tris pH 6.8 and 160 mM DTT), and the protein concentration was determined.

B. Determination of protein concentration
Bradford Coomassie Blue method was used. Bovine Serum Albumin (BSA, 2 mg / ml) was used as a standard substance.
1. Stock solution BSA was diluted to 1 mg / ml to prepare a BSA standard concentration series solution (containing 0,1,5,10,15,20,25 μg BSA in 500 μl deionized water (d-H 2 O)).
2. 1 μl SDS sample buffer was added to the BSA standard series solution.
3. Samples were prepared by adding 1 μl protein lysate to 500 μl d-H 2 O.
4. 500 μl Coomassie Blue reagent was added and mixed. To prevent precipitation, the mixed solution must not be left too long at room temperature.
5. Absorbance was 595 nm. The cell lysate was stored at -20 ^° C, and if the total volume was large, 0.5 ml was divided into Eppendorf tubes.

C. SDS-PAGE Separation Gel 1.15 ml of resolving gel was prepared in the flask and kept stirring for 15 minutes.
10w / w% SDS resolving gel:
30w / w% Acrylamide / Bis 4.95ml
1.5M Tris-HCl, pH8.8 3.75ml
10w / w% SDS 0.15ml
d-H2O 6ml
The following solutions were added before pouring the separation gel between the plates.
TEMED (N, N, N, N-Tetramethylethylenediamine) 7.5μl
10w / w% APS (Ammonium Persulfate) 75μl
2. Gloves were attached, the plate was washed with 95 v / v% ethanol, wiped with Kimwipe and dried. The plate was assembled and stood on the plate holder. A plate with a shorter plate length was directed outward.
3. TEMED and 10 w / w% APS were added to the separated gel solution and stirred for a short time. Using a pipette, the solution was added to the gap between the plates up to 0.5 cm from the top of the shorter plate. Immediately, a small amount of butanol saturated with water was added to cover the top of the separation gel.
4). Care was taken to prevent leakage from the bottom of the plate, and the plate was left on the bench for 1 hour.
5. Butanol was removed and the surface of the gel was washed with dH ₂ O and dried with Kimwipe.
6.5 ml of a stacking gel was prepared.
4w / w% SDS stacking gel:
30w / w% Acrylamide / Bis 0.66ml
1M Tris-HCl, pH6.8 1.28ml
10w / w% SDS 50μl
dH ₂ O 3ml
The following solutions were added before pouring the concentrated gel.
TEMED 5μl
10w / w% APS 25μl
7). The concentrated gel was poured onto the top of the separating gel to the top of the short plate and immediately the sample comb was inserted into the gap between the plates.
8). Let stand for at least 5 hours to polymerize.
9. After carefully removing the comb and rinsing the surface of the gel with d-H2O, the gel was dried.
10. A 500 ml running buffer (25 mM Tris-HCl, 0.2 M glycine, 0.35 w / w% SDS) was prepared.
11. The gel was clamped to the electrophoresis holder and placed in the tank. The electrophoresis buffer was poured between the two plates to the top of the long plate. Care was taken not to have a buffer from the upper chamber to the bottom.
12 The protein lysate was mixed with the same volume of 2x loading buffer (62.5 mM Tris HCL, pH 6.8, 25 w / w% Glycerol, 2 w / w% SDS, 5 w / w% Mercaptoethanol and 0.01 w / w% Bromopohenol Blue).
13. Lysate (5 μg / well) was poured in two lanes.
14 Electrophoresis was performed at 150 V for 1 hour until the dye reached the bottom of the gel.

D. Semi-dry transfer buffer: 60mM Tris, 40mM Glycine and 15v / v% Methanol (pH9.2)
1. The gel was immersed in 200 ml of transfer buffer for 30 minutes.
2. The PVDF membrane was wetted in methanol for 15 seconds, rinsed with deionized water, and then immersed in the transfer buffer for at least 15 minutes. Two filter papers were soaked in transfer buffer for 15 minutes.
3. The plate was assembled in the order of + pole side filter paper, membrane, gel, and -pole side filter paper from the + pole side to the -pole side. The membrane and filter paper were cut to the same size as the gel, and care was taken not to generate bubbles between the membrane and the gel.
4. Power was applied at 15-20V for 30-45 minutes (power and time can be adjusted by gel size).

E. Immunoblotting The cassette was opened and the gel was removed from the transfer PVDF membrane.
2. The transfer membrane was placed in a blocking buffer (20 mM Tris Base pH 7.6, 150 mM NaCl, 0.1 w / w% Tween-20, 5 w / w% non-fat milk) and incubated at room temperature for 1 hour.
3. The membrane was removed from the blocking buffer and rinsed with a wash buffer (20 mM Tris Base pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 30 minutes. The rinsing was performed while changing the washing buffer every 10 minutes and stirring.
4). After blocking, the membrane was incubated with the primary antibody while gently seeking overnight at 4 ° C. in 5% non fat milk.
5. After rinsing, the primary antibody buffer was decanted from the membrane and the membrane was rinsed with wash buffer for 30 minutes. Rinse was performed while changing the wash buffer every 10 minutes and stirring.
6). Secondary antibody (Horseradish peroxidase-conjugated antibody) was incubated with block buffer (secondary antibody: diluted so that block buffer was 1: 50,000) and incubated at room temperature for 1-2 hours.
7). The secondary antibody buffer was removed and the membrane was rinsed with wash buffer for 30 minutes. Rinse was performed while changing the wash buffer every 10 minutes and stirring.

F. Band detection ECL (Enhanced Chemiluminescence) reagent (same volume mixture of solution A and solution B (GE Healthcare, UK)) was prepared.
2. The reagent was added to the membrane and allowed to react for 5 minutes.
3. The stained membrane was measured with VersaDoc 5000, and the band pattern was analyzed using Quantity One software.

  The results of Western blotting analysis of mixed breast tissue samples (Mixed Breast Tissue: a mixture of breast tissue extracts from 3 breast cancer patients and a mixture of breast tissue extracts from 6 normal persons) are shown in FIGS. 11 shows. In each figure, the results of Western blotting for breast cancer markers in a breast cancer sample (ER +) and a normal sample (TNT) using an actin antibody (Anti-actin) as a reference are shown in (A). Also, a graph showing the integrated value (calculated by PDQuest software) of the absorbance of the antibody band of the breast cancer marker in (A) is shown in (B).

[Conclusion about the biomarker of the present invention]
As described above, CRAB, 6PGL and CAZ2 were found to be breast cancer biomarkers based on tissue microarray analysis and validation by Western blotting. (Note that ANX1 is also found to be a breast cancer biomarker. ANX1 has already been shown by previous literature to be a breast cancer marker.) CRAB shows down-regulation in breast cancer Biomarkers, 6PGL and CAZ2 are biomarkers that show upregulation in breast cancer.

2 shows a flow of a marker search method used for breast cancer marker search of the present invention. The result of the gene collection enrichment analysis (GSEA) of the protein which shows the different expression in mRNA expression data is shown. (A): relates to a protein identified by comparison between breast cancer cell line MCF-7 and normal cell line CCD-1059Sk. (B): relates to a protein identified by comparison between breast cancer cell line HCC-38 and normal cell line CCD-1059Sk. The result of the principal component analysis (PCA) of mRNA expression about nine types of breast cancer marker candidates (ANX1, CRAB, 6PGL, CAZ2, K2C7, LAM1, TKT, PAK2, and PSD2) is shown. It is the result of investigating the intracellular localization of breast cancer markers 6PGL and CAZ2 by Western blotting. A breast tissue (magnification: × 100) subjected to tissue microarray analysis (TMA) for the breast cancer marker ANX1 is shown together with a partial enlarged view (magnification: × 600) of the tissue (N: normal tissue, T: breast cancer tissue). A breast tissue (magnification: × 100) subjected to tissue microarray analysis (TMA) for the breast cancer marker CRAB is shown together with a partial enlarged view (magnification: × 600) of the tissue (N: normal tissue, T: breast cancer tissue). A breast tissue (magnification: × 100) subjected to tissue microarray analysis (TMA) for the breast cancer marker 6PGL is shown together with a partial enlarged view (magnification: × 600) of the tissue (N: normal tissue, T: breast cancer tissue). A breast tissue (magnification: × 100) subjected to tissue microarray analysis (TMA) for the breast cancer marker CAZ2 is shown together with an enlarged view (magnification: × 600) of a part of the tissue (N: normal tissue, T: breast cancer tissue). (A): Western blotting results for breast cancer marker CRAB in breast cancer sample (ER +) and normal sample (TNT) using actin antibody (Anti-actin) as a reference, and (B): breast cancer in (A) It is the graph which showed the integrated value of the light absorbency of the band of the marker antibody (Anti-CRAB). (A): Results of Western blotting for breast cancer marker 6PGL in breast cancer sample (ER +) and normal sample (TNT) using actin antibody (Anti-actin) as a reference, and (B): breast cancer in (A) It is the graph which showed the integrated value of the light absorbency of the band of the antibody (Anti-6PGL) of a marker. (A): Results of Western blotting for breast cancer marker CAZ2 in breast cancer sample (ER +) and normal sample (TNT) using actin antibody (Anti-actin) as a reference, and (B): breast cancer in (A) It is the graph which showed the integrated value of the light absorbency of the band of the antibody (Anti-CAZ2) of a marker.

Claims (6)

  Alpha Basic—Estrogen receptor positive breast cancer marker, including crystallin.   A breast cancer marker comprising 6-phosphogluconolactonase and / or F-actin capping protein alpha-2 subunit.   Alpha Basic-A method for identifying breast cancer morbidity by using crystallin as a protein of an estrogen receptor positive breast cancer marker. Measuring the level of said protein in a sample from an individual to whom breast cancer affliction is to be identified,
Comparing the measured level obtained with the normal level of the protein,
The obtained measured value level is decreased from the normal level as an indicator that the subject individual is likely to have estrogen receptor-positive breast cancer. 4. A method for identifying the incidence of breast cancer according to 3. A method for identifying breast cancer morbidity by using 6-phosphogluconolactonase and / or F-actin capping protein alpha-2 subunit as a protein for breast cancer markers. Measuring the level of said protein in a sample from an individual to whom breast cancer affliction is to be identified,
Comparing the measured level obtained with the normal level of the protein,
The obtained measured value level is higher than the normal level, which is one of the indicators that the subject individual is likely to have breast cancer. A method of identifying the incidence of breast cancer.