JP2007175023A - Composition and method for predicting prognosis and metastasis risk of cancer patient after operation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and composition for the in vitro prediction of the prognosis or metastasis risk of a cancer patient after operation by using a cancer metastasis relating gene group as a marker, and provide a method for screening a cancer metastasis suppressing agent on the inhibition or suppression of a cancer metastasis relating gene or its transcription product. <P>SOLUTION: The invention provides a method for the in vitro prediction of the prognosis or the metastasis risk of a cancer patient after operation. At least one expression level of a cancer metastasis relating gene marker in a biological specimen derived from a patient or its transcription or translation product is determined by using a probe corresponding to the marker, and the prognosis or the metastasis risk of a patient after operation is judged by using an index comprising the relative level difference between a patient group having better prognosis or lower metastasis risk after operation and a patient group having worse prognosis or higher metastasis risk. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a composition or method for predicting postoperative prognosis or metastatic potential in cancer patients in vitro.

  The present invention also relates to a method for screening a cancer metastasis inhibitor using cultured cancer cells.

  Cancer is a multifactorial disease in which genes and environmental factors are complicatedly involved. Various therapies such as chemotherapy, radiotherapy, surgery, and immunotherapy have been implemented, but due to the complexity of the onset mechanism of cancer, it is not always effective enough for the treatment of middle-stage and end-stage cancer and suppression of cancer metastasis. Is not achieved.

  On the other hand, it is also true that many cancer patients are cured or prolong their lives by early detection and early treatment of cancer. For cancer diagnosis, methods such as cytology, histology, endoscope, image diagnosis, and biochemical diagnosis using cancer markers are adopted. It is active.

  One of the major causes that hinders cancer treatment and recurrence is cancer metastasis. Due to the motility and invasion ability of cancer, cancer metastasizes from the primary focus to another tissue and grows. The overall picture of this transition mechanism is largely unknown. As cancers become more malignant and when cancer is removed, cancers are more likely to metastasize, resulting in cancer recurrence. Preventing cancer metastasis is accompanied by the development of early diagnosis and effective treatment of cancer. It has become an important issue in the medical field.

  At present, several cancer metastasis-related genes have been reported, most of which have been discovered by the differential display method. This method detects target genes based on differences in expression patterns, but many of the genes found by this method are genes that are commonly expressed in a certain amount in many tissues and cells, so-called househouses. It is a keeping gene. If genes that are essentially involved in cancer metastasis can be identified, it is expected to lead to the development of drugs that suppress cancer metastasis.

  As a cancer metastasis inhibitor, for example, a substance that inhibits signal transduction via human VEGF receptor Flt-1 (Patent Document 1), a peptide having cell adhesion inhibitory activity (Patent Document 2), and a purine system having a purine receptor working function A compound (Patent Document 3) and the like are known. In addition, studies on genes involved in cancer metastasis suggest that matrix degrading enzymes, vascular endothelial growth factors and their receptors are involved in metastasis. For example, a cancer metastasis-related gene discovered from a mouse cell line has been reported as a cancer metastasis-related gene (Patent Document 4).

  In recent years, application of gene suppression technology targeting mRNA to cancer treatment is being attempted. Such gene suppression techniques include, for example, antisense methods, ribozymes, RNA interference (RNAi), and the like (Non-patent Document 1). In particular, since RNAi has high specificity, basic research for its medical application is rapidly progressing, and attention is focused on viral diseases, cancers, genetic diseases and the like as its targets (non-patented) Reference 2).

JP2003-261460 JP 09-143076 A Japanese Patent Laid-Open No. 7-082156 Table 98/045431 Daisuke Seki et al., Experimental Medicine Vol.22, No.14 (Special Issue), 89-98, 2004, Yodosha Experimental Medicine Vol.22, No.4 "Science of RNAi", 2004, Yodosha

  So far, it has been suggested that a certain gene or polypeptide may be related to cancer metastasis, but at the laboratory level it is related to metastasis, but other systems confirm that it is related to metastasis. There were many events that could not be achieved or that the development of inhibitors and clinical application did not provide sufficient effects.

  In such a situation, the present inventors focused on the fact that metastasis is not caused by a single gene but by a coordinated interaction of a plurality of gene groups. The relationship between genetic markers and postoperative prognosis or metastatic potential of cancer patients was examined.

  An object of the present invention is to provide a method and a composition for predicting the postoperative prognosis or metastasis potential of cancer patients in vitro using cancer metastasis-related genes as markers.

  Another object of the present invention is to provide a method for screening a cancer metastasis inhibitor for inhibition or suppression of a cancer metastasis-related gene or a transcription product thereof.

  Genes that show differential expression between the high metastatic human lung cancer cell line developed by the present inventors and their low metastatic parental lines, and that are considered to be involved in acquiring metastatic potential. We identified groups and further investigated the possibility of metastasis and recurrence in human cancer patients using comprehensive gene expression data collected using different microarray platforms for multiple carcinomas. As a result, in several carcinomas, the cancer patient group is roughly divided into two groups according to the expression profile of the identified gene group, and there is a significant difference in survival time (or prognosis) after surgery between the two groups. Was found to indicate the presence of.

SUMMARY OF THE INVENTION In summary, the present invention has the following characteristics.
(1) A method for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, wherein the nucleotide sequence shown in SEQ ID NOs: 1 to 45 or a mutant sequence thereof in a biological sample derived from the patient A group of patients having a better prognosis after surgery or a lower possibility of metastasis, by measuring at least one expression level of a cancer metastasis-related gene marker or a transcription or translation product level thereof using a probe corresponding to the marker; The method as described above, comprising determining the postoperative prognosis or metastatic potential using the relative difference in level between a group of patients with a worse postoperative prognosis or a higher possibility of metastasis as an index.
(2) When the cancer metastasis-related gene marker comprises the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient is identified as a patient with a better prognosis after surgery or a lower possibility of metastasis. The method according to (1) above.
(3) When the cancer metastasis-related gene marker is composed of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, the patient is identified as a patient with a worse prognosis after surgery or a higher possibility of metastasis. The method according to (1) above.
(4) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1-45, a polynucleotide complementary thereto, a variant thereof, a polynucleotide that hybridizes to them under stringent conditions, or 15 The method according to any one of (1) to (3) above, which is selected from the group consisting of the above-mentioned fragments containing consecutive bases.
(5) The probe is an antibody or a fragment thereof against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof. The method according to any one of (1) to (3) above.

(6) The method according to (5) above, wherein the antibody is a polyclonal antibody, a monoclonal antibody or a peptide antibody.
(7) The method according to any one of (1) to (6), wherein the cancer is a solid cancer.
(8) The method according to (7) above, wherein the solid cancer is lung cancer or breast cancer.
(9) A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1-45, a complementary polynucleotide thereto, a variant thereof, a polynucleotide that hybridizes under stringent conditions thereto, or 15 or more consecutive nucleotides A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising at least one probe selected from the group consisting of those fragments containing bases.
(10) A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 28 to 45, a polynucleotide complementary thereto, a variant thereof, a polynucleotide that hybridizes under stringent conditions thereto, or 15 or more consecutive nucleotides A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising at least one probe selected from the group consisting of those fragments containing bases.

(11) A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 27, a polynucleotide complementary thereto, a variant thereof, a polynucleotide that hybridizes to them under stringent conditions, or 15 or more consecutive nucleotides A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising at least one probe selected from the group consisting of those fragments containing bases.
(12) selected from the group consisting of an antibody against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof; A composition for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, comprising at least one probe.
(13) selected from the group consisting of an antibody against a polypeptide selected from the group consisting of a polypeptide consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a polypeptide encoded by a variant thereof, or a fragment thereof; A composition for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, comprising at least one probe.
(14) selected from the group consisting of an antibody or a fragment thereof against a polypeptide selected from the group consisting of a polypeptide consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a variant thereof, or a fragment thereof A composition for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, comprising at least one probe.
(15) The composition according to any one of (9) to (14), wherein the probe is included in the form of a kit.

(16) The composition according to any one of (9) to (11), wherein the probe is included in the form of a microarray.
(17) Inhibition or suppression of a cancer metastasis-related gene comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 27 or a transcription product thereof using cultured cancer cells, or the motility and / or invasive ability of human cultured cancer cells A screening method for a cancer metastasis inhibitor comprising screening a candidate drug for inhibition or suppression of cancer.
(18) Preparing cultured cancer cells, culturing the cells in the presence of a candidate drug, and inhibiting or suppressing the expression of the cancer metastasis-related gene or its transcription product, or the motility and / or invasion of the cultured cancer cells The method according to (17) above, comprising screening a candidate drug for inhibition or suppression of ability.

  The present inventors have found that 45 types of cancer metastasis-related gene markers identified this time are very useful in determining the postoperative prognosis or metastatic potential of cancer patients in a plurality of carcinomas. It was. By using these markers as indicators, it is possible to predict the postoperative prognosis of cancer patients and the possibility of cancer metastasis, so that treatment planning for patients, improvement of treatment results for cancer, and recurrence due to metastatic cancer can be achieved. It can be expected to be of great help for prevention and prognosis management. Furthermore, in cultured cancer cells, it is possible to screen for an agent that regulates its expression using the marker as an indicator, thereby leading to the development of a drug that suppresses or prevents cancer metastasis.

Definitions The terms used in this specification have the following meanings.
As used herein, “postoperative prognosis” means determining the prognosis of a patient after surgery for cancer, typically by the survival rate at 5 years postoperatively. Prognosis is closely related to cancer metastasis, and “poor prognosis” means that the cancer is highly invasive, metastatic, or advanced, whereas “good prognosis” means that Meaning less invasive, metastatic or less advanced.

  As used herein, “metastasis” refers to a series of processes by which cancer cells, by virtue of their motility and invasive potential, migrate from the primary focus to distant tissue where they proliferate and form a neoplasm. . Metastasis is a major obstacle to cancer treatment because it causes cancer recurrence. It is also known that cancer cells infiltrating into blood vessels or lymph vessels stay in various tissues and cause multiple cancer diseases. In the present invention, any metastasis is targeted as long as the cancer metastasis-related gene marker of the present invention is involved. For example, lymph node metastasis can also be targeted.

  As used herein, “patient” refers to mammals including humans, dogs and cats, with preferred mammals being humans.

  As used herein, a “biological specimen” refers to a tissue, cell or fluid collected from a mammal (preferably a human), preferably a cancer tissue or cell. In the present invention, cancer tissue or cells are derived from a patient group having a better postoperative prognosis or a lower possibility of metastasis, and from a patient group having a lower postoperative prognosis or a higher possibility of metastasis. Any cancer can be used as long as it shows a relative difference in the expression level of the gene marker related to the present invention. Examples of cancer include lung cancer, breast cancer, colon cancer, prostate cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, kidney cancer, uterine body / cervical cancer, ovarian cancer, bladder cancer, brain tumor, thyroid cancer, lymphoma, Examples include, but are not limited to, testicular cancer, osteosarcoma, skin cancer, melanoma, blood cancer (particularly leukemia), and the like.

  As used herein, a “cancer metastasis-related gene marker” has now been found by identifying genes that differ in expression levels between high and low metastatic cancer cell lines. It is a marker and can be used to determine the postoperative prognosis or metastatic potential of cancer patients as in the method of the present invention.

  As used herein, “variant” refers to, for example, a mutant caused by biological events such as mutation, polymorphism, alternative splicing, degeneracy of the genetic code, homologs between species, etc. Is included. The mutant includes one or more, preferably one or several substitutions on the sequence of the gene containing the nucleotide sequence represented by SEQ ID NOs: 1 to 45 related to the present invention, or the polypeptide encoded by the gene. , Mutants having mutations such as deletion, addition, insertion, etc., mutations comprising the sequence or a partial sequence thereof having a sequence having 80%, 85%, 90%, 95%, 98% or more identity Body, etc. are included. Here, “several” usually means an integer of 10 or less. In addition, the identity (%) can be determined using a known BLAST or FASTA program into which a gap is introduced (SF Altschul et al., J. Mol. Bio. 215: 403-410, 1990). In general, identity (%) can be calculated as a percentage of the number of matched bases relative to the total number of bases.

  As used herein, “stringent conditions” are not limited to the following, but hybridization and wash conditions such that nucleotide sequences having at least 80%, preferably at least 95% identity, hybridize to each other: For example, hybridization and washing conditions in microarray analysis are hybridization in 1 M sodium chloride / 0.5% (W / V) sarkosyl / 30% formamide at 60 ° C. for 17 hours, followed by 6 × SSC / Once in a 0.005% (W / V) Triton X-102 solution at room temperature, once for 10 minutes, and further in a 0.1 × SSC / 0.005% (W / V) Triton X-102 solution This is a condition of washing once in 5 minutes while keeping at 4 ° C. Here, 1 × SSC is 150 mM sodium chloride and 15 mM sodium citrate aqueous solution (pH 7.2). For hybridization, see F.C. M.M. Ausbel et al., Short Protocols in Molecular Biology (3rd edition) A Compendium of Methods Current Protocols in Molecular Biology, 1995, John Wisley. (United States).

Detailed Description In the following, the present invention will be described in more detail.
1. Cancer metastasis-related gene marker The human cancer metastasis-related gene marker of the present invention was found as follows.
Based on subculture of cells from lung metastases and metastatic ability after transplanting subcutaneously to mice with known metastatic lung cancer cell line NCI-H460 (American Type Culture Collection) (parent strain) in mice The highly metastatic cancer cell line LNM35 was isolated by in vivo selection using mice (see FIG. 1). On the other hand, a cancer cell line with further low metastasis can be obtained from NCI-H460 cells by treatment with a limiting dilution method or a demethylating agent. In the cultured cell or mouse transplantation test, the LNM35 strain showed a very high value compared to the parent strain or the low-metastatic cancer cell line in any of motility, invasive ability, lymph node metastasis, and lung metastasis (see FIG. 2). .

  By examining the gene expression difference by gene filter microarray (Research Genetics) between the high-metastasis cancer cell line LNM35 and the parent or low-metastasis cancer cell line, the nucleotide sequences of SEQ ID NOs: 1 to 45 were obtained. Including human cancer metastasis-related genes. As a result of the data bank search, these genes are registered in Unigene or GenBank as shown in Table 1, but their relevance to cancer metastasis is not known. Table 1 and Table 2 respectively show the sequence number, GenBank accession number, UniGene ID, and gene name (Gene symbol, Gene name) of the gene marker having high expression and low expression in the highly metastatic LNM35 strain. Indicates.

  The cancer metastasis-related marker identified as described above can be a marker not only in lung cancer but also in solid cancer such as breast cancer. While the markers reported so far have been specific for certain cancers, it is surprising that the markers of the present invention are common markers for various cancers.

  For this reason, for example, lung cancer, breast cancer, colon cancer, prostate cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, kidney cancer, uterine body / cervical cancer, Solid cancers such as ovarian cancer, bladder cancer, brain tumor, thyroid cancer, lymphoma, testicular cancer, osteosarcoma, skin cancer, melanoma are included. Further possible cancers include blood cancer (particularly leukemia).

  In the present invention, cancer metastasis-related gene markers include not only genes containing the nucleotide sequences shown in SEQ ID NOs: 1-45 but also mutant genes thereof. Usually, such variants include those that occur in vivo as a result of biological events such as mutations, polymorphisms, alternative splicing, and the like. In practice, the genetic marker is detected at the nucleic acid or protein level as a transcript or translation product.

  In the case of a transcript, it is detected as mRNA, cRNA or cDNA. Total RNA is extracted from cells or tissues by a method such as the phenol / chloroform method, poly A (+) RNA or mRNA is prepared by the oligo dT cellulose column method, and if necessary, cDNA and cRNA are further synthesized from the mRNA.

  In the case of a translation product, it is detected as a protein or fragment thereof encoded by the gene or variant thereof.

  The cancer-related gene markers according to the present invention are roughly classified into two groups according to their expression patterns, corresponding to the prognosis of cancer patients after surgery or the degree of metastasis (see FIGS. 3 to 5).

  Specifically, when the cancer metastasis-related gene marker consists of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient is identified as a patient with a better prognosis after surgery or a lower possibility of metastasis. Is done. On the other hand, when the cancer metastasis-related gene marker consists of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, the patient is identified as a patient with a worse prognosis after surgery or a higher possibility of metastasis.

2. Composition for Predicting Prognosis or Metastatic Potential As described above, the present invention provides a method for predicting the postoperative prognosis or metastatic potential of a cancer patient in vitro, and this method comprises an organism derived from the patient. Using a probe corresponding to the expression level or transcription or translation product level of at least one cancer metastasis-related gene marker comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45 or a mutant sequence thereof in a biological specimen And predicting postoperative prognosis or metastatic potential using as an index a significant level difference with respect to a normal or non-cancerous control sample.

  The probe used in the present invention is not particularly limited as long as the marker can be detected, but is usually a polynucleotide or an antibody. Accordingly, compositions comprising such polynucleotides or antibodies can be used to predict postoperative prognosis or metastatic potential of cancer patients.

2.1 Polynucleotide Probe Specifically, the polynucleotide probe according to the present invention is (i) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1 to 45, and (ii) complementary to the polynucleotide of (i). A polynucleotide of (iii) (i) and (ii), a polynucleotide of (iv) (i) or (ii) or a variant of (iii) under stringent conditions And (v) at least one, preferably at least 2, more preferably 3 to 45, selected from the group consisting of a fragment comprising 15 or more consecutive bases of said polynucleotide or variant, for example, Includes 4 to 45, 5 to 45 probes.

  The cancer metastasis-related gene markers according to the present invention can be divided into two groups as follows according to the prognosis status (survival rate) of cancer patients.

  That is, Group 1 is a case where the postoperative prognosis of cancer patients is worse, and the markers involved in this group are the gene groups of SEQ ID NOs: 1 to 27, while Group 2 is the postoperative prognosis of cancer patients. The markers involved in this group are the gene groups of SEQ ID NOs: 28-45.

  As used herein, “good prognosis” means that the cancer is highly invasive, metastatic, or advanced, and the proportion of 5-year survivors of cancer patients determined in this way is about 70%. Whereas “prognosis is poor” means that the cancer is less invasive, metastatic, or less advanced, and the proportion of 5-year survivors of cancer patients is about 50% or less .

In the present invention, probes corresponding to at least one, preferably at least 2, more preferably 3 to all, for example, 4 to all, and 5 to all markers among the gene markers belonging to each group. Use to inspect.
Probes for testing markers belonging to Group 1 include polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 27, polynucleotides complementary thereto, variants thereof, and hybrids under stringent conditions to them. Probes selected from the group consisting of soy polynucleotides or fragments thereof containing 15 or more contiguous bases are included.

  On the other hand, a probe for examining a marker belonging to Group 2 includes a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 28 to 45, a complementary polynucleotide thereto, a variant thereof, and a stringent condition thereto. Or a probe selected from the group consisting of fragments thereof comprising 15 or more consecutive bases.

  Thus, the present invention also includes a composition including each probe group related to these groups 1 and 2.

  In the present invention, a mutant as a probe is a mutation such as substitution, deletion, addition or insertion of one or more, preferably one or several, in the base sequence represented by SEQ ID NOs: 1 to 45 as defined above. And mutants composed of sequences having the identity of usually 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more and 98% or more.

  Furthermore, in the present invention, the polynucleotide that hybridizes under stringent conditions may hybridize with the polynucleotide or variant of (i) to (iii) above under the hybridization conditions as defined above. If possible, there is no particular limitation. Such a polynucleotide may have a gene in which the nucleotide sequence represented by SEQ ID NOs: 1 to 45 is changed due to biological events such as mutation, polymorphism, and alternative splicing depending on individual patients. Allows detection of the gene.

  Furthermore, in the present invention, the fragment of the polynucleotide has a size of 15 bases to less than the total bases. The fragment has any number of bases within this range, for example, 20 bases or more, 30 bases or more, 50 bases or more, 70 bases or more, 100 bases or more, 150 bases or more, 200 bases or more, 250 bases or more.

  The polynucleotide probe used in the present invention can be synthesized by a conventional chemical DNA synthesis technique or a gene recombination technique.

  If the polynucleotide is a DNA molecule of about 100 bases or less, it can be synthesized using an automatic DNA synthesizer (for example, Applied Biosystems, USA) using the phosphoramidite method.

  Alternatively, the polynucleotide can be produced by cDNA cloning. After obtaining total RNA from the target cancer tissue and obtaining poly A (+) RNA by oligo dT cellulose column treatment, a cDNA library was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) method. From the library, probes (15 or more, preferably 30 or more, more preferably 50 to 100 or more) prepared in advance based on sequences (Table 1 and Table 2) registered in data banks such as GenBank and UniGene. CDNA clones can be obtained by hybridization with (base length). The obtained clone is incorporated into an expression vector such as a commercially available one, introduced into an appropriate host cell such as Escherichia coli or Bacillus subtilis, and propagated, or based on a sequence registered in the data bank. Amplification can be performed by polymerase chain reaction (PCR) using a primer prepared in advance (usually 15 to 30 bases, preferably 17 to 25 bases long) and using the cDNA clone as a template. For specific procedures and reagents for cDNA cloning and PCR, commercially available kits, devices, and reagents can be used. For example, Sambrook J et al., Molecular Cloning A Laboratory Manual, 1989, Cold Spring Harbor Laboratory. Press (USA); M.M. Ausbel et al., Short Protocols in Molecular Biology (3rd edition) A Compendium of Methods Current Protocols in Molecular Biology, 1995, John Wisley. (United States).

2.2 Antibody Probe In the present invention, an antibody may be used as a probe. Examples of antibodies include the following.
(I) an antibody or a fragment thereof against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof;
(Ii) an antibody or a fragment thereof against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a polypeptide encoded by a variant thereof, or a fragment thereof, and
(Iii) An antibody or fragment thereof against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a polypeptide encoded by a variant thereof, or a fragment thereof.

  Accordingly, the present invention provides an antibody or fragment thereof described in (i), (ii) and (iii) above, preferably at least one selected from each of (ii) and (iii), preferably at least 2 And more preferably 3 to all, for example 4 to all, 5 to all antibodies or fragments thereof.

The antibodies used in the present invention include polyclonal antibodies, monoclonal antibodies, anti-peptide antibodies and the like. Antibody fragments include Fab, (Fab ′) ₂ , Fc, Fc ′, Fd, Fv, and the like. These antibody fragments can be obtained, for example, by limited degradation of the antibody with a protease such as papain or pepsin.

  Polypeptides or fragments thereof corresponding to each gene are synthesized using protein synthesis or gene recombination techniques, and the resulting polypeptides or fragments thereof are used as antigens for rabbits, mice, rats, horses, cows, goats, Animals such as sheep are immunized and antibodies against their antigens are produced and purified.

  The polyclonal antibody is obtained by immunizing the animal subcutaneously with about 10 to 300 μg of the antigen, further immunizing about 2 weeks later, collecting blood about 3 weeks to 1 month after the initial immunization, and obtaining the desired polyclonal antibody from the antiserum. The IgG component can be produced by a method including separation using ammonium sulfate fractionation and ion exchange chromatography. In order to increase specificity, the obtained IgG is bound to a column made by binding the target protein to a carrier such as cellulose or agarose, and then eluted with a high salt buffer for dialysis or ultrafiltration. A specific polyclonal antibody can be obtained by desalting by a method such as The antibody titer can be measured by a conventional immunoassay, for example, an enzyme immunoassay (EIA, ELISA), a radioimmunoassay (RIA), a fluorescent antibody method, or the like.

A monoclonal antibody can be prepared, for example, by the following general method.
The target polypeptide or a fragment thereof is administered subcutaneously to mice or rats (for example, Balb / c mice) in the same manner as in the production of polyclonal antibodies, and booster immunization is performed about 2 to 4 times at intervals of 1 to 4 weeks. When the antibody titer reaches a peak, the antigen is injected intravenously or intraperitoneally to obtain the final immunization. After 2 to 5 days, antibody-producing cells (eg spleen cells or lymph node cells) are collected. The antibody-producing cells are then fused to a myeloma cell line (preferably a hypoxanthine, guanine, phosphoribosyl transferase (HGPRT) deficient cell line) to generate hybridoma cells, and HAT (hypoxanthine, aminopterin, thymine) selection is performed. Do. For cell fusion, antibody-producing cells and myeloma cell lines are mixed in a ratio of about 1: 1 to 20: 1 in animal cell culture media such as serum-free DMEM, RPMI-1640 medium, and the like. It is carried out in the presence of a cell fusion promoter such as Confirmation of the target antibody can be performed by the immunoassay described above. Furthermore, for the growth of hybridomas, about 10 million hybridomas are administered into the abdominal cavity of mice to allow the hybridomas to grow, and ascites is collected after 1 to 2 weeks. Antibody purification can be performed by appropriately combining methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel chromatography.

  Anti-peptide antibodies are antibodies against linear peptides on the surface of proteins and can increase immunological specificity. Such peptides include, for example, the estimation of hydrophilic-hydrophobic regions by Kyte-Doolittle, et al., The probability of being located on the surface of a specific peptide site on a protein molecule by Emini et al., The degree of bending of a polypeptide chain, such as Chou-Fasman Can be estimated using an α helix, β sheet, secondary structure prediction of a protein indicating a turn, etc. alone or in combination. The estimated peptide can then be synthesized using a peptide synthesizer.

  Here, the synthesis of the target polypeptide (encoded by the genes shown in Tables 1 and 2 above) is achieved by incorporating a cDNA clone into an expression vector, and a prokaryotic or eukaryotic host cell transformed or transfected with the vector. Can be obtained from the cells or the culture supernatant. A commercially available expression vector can be used. Host cells include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis, Pseudomonas bacteria), yeast (eg, Saccharomyces, Pichia, etc.), insect cells (eg, Sf cells), mammalian cells (eg, CHO, COS, BHK, HEK293, etc.). The vector is composed of a plasmid, cosmid, phage or the like, and can contain DNA encoding the target polypeptide, promoter, enhancer, polyadenylation signal, ribosome binding site, replication origin, terminator, selectable marker, etc. if necessary. . To facilitate polypeptide purification, a DNA sequence encoding a labeled peptide, such as a 6-10 residue histidine tag, FLAG, GFP polypeptide, etc., can also be included. The gene recombination techniques are described in Sambrook et al. (Above), Ausbel et al. (Above), and the techniques described therein can be used for the present invention.

  The target polypeptide obtained as described above is appropriately subjected to gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, isoelectric focusing, electrophoresis, ultrafiltration, salting out, dialysis, etc. It can be purified in combination.

  The sequence of the target polypeptide or fragment thereof can be obtained by accessing the NCBI HomePage based on the GenBank or UniGene accession numbers listed in Tables 1 and 2 above.

  According to an embodiment of the present invention, the composition may be in the form of a kit or microarray, i.e., the probe is included in the form of a kit or microarray.

  In the case of a kit, a polynucleotide or an antibody capable of detecting the total number of markers from one or more of each gene marker of two groups (Table 1 and Table 2), individually or in combination of two or more, is suitable. Can be packaged in a container.

The antibody is a polyclonal antibody, a monoclonal antibody, an anti-peptide antibody or the like as prepared by the above method, but is not limited thereto. The type of antibody may be any type, class, subclass, and includes, for example, IgG, IgM, IgE, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2. Antibody fragments include Fab, (Fab ′) ₂ , Fc, Fd, Fv, and the like.

  The kit may further contain reagents for performing hybridization, such as a buffer, a reverse transcriptase, a labeled secondary antibody, and the like.

  In the case of a microarray, the array is a DNA microarray (also referred to as a DNA chip), a tissue array, or a protein microarray.

  Each of these microarrays is bound with the above-described polynucleotide as a probe or the above-described antibody or fragment thereof. That is, on the surface of the array, a polynucleotide capable of detecting the gene marker or transcription or translation product, a polynucleotide capable of hybridizing with the gene marker or a variant thereof, or a polypeptide encoded by these genes, Alternatively, an antibody or a fragment thereof that can specifically bind to the mutant or derivative thereof is bound as a probe.

  As the substrate of the array, glass or resin (polymer) is usually used, and poly L-lysine, silane or densified amino groups are introduced on the surface thereof. The polynucleotide or antibody is bound to the substrate by a spot method or an ink jet method.

  Mutants generally have 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, 98% or more identity with the complete or partial sequence of the gene or polypeptide at the nucleotide or amino acid level. I have it.

  Derivatives of polypeptides include, for example, chemically modified derivatives such as glycosylation, phosphorylation, sulfation, alkylation, acylation and the like.

3. Method for Predicting Prognosis or Metastasis Potential The method of the present invention is a method for predicting the postoperative prognosis or metastasis potential of cancer patients in vitro, and comprising SEQ ID NO: 1 in a biological specimen derived from the patient. The expression level of at least one cancer metastasis-related gene marker comprising the nucleotide sequence shown in -45 or a mutant sequence thereof or the transcription or translation product level thereof is measured using a probe corresponding to the marker, and the prognosis after surgery is determined. Postoperative prognosis or metastasis, using the relative difference in level between better or less likely metastatic patients and worse postoperative prognosis or more likely metastasis Including determining the possibility.

  Here, the expression level is the transcription or translation product level for the expression of the above gene in a biological specimen, and a specimen from a patient group with a better postoperative prognosis or a lower possibility of metastasis; It means the difference in the expression of the gene when comparing the expression of the gene with specimens from a patient group with a worse prognosis or a higher probability of metastasis.

  By identifying the above genes that show differential expression levels, the postoperative prognosis or metastatic potential of cancer patients can be predicted in vitro.

  According to the present invention, the difference in the expression level of a gene can be performed by measuring the presence or amount of the gene or a polypeptide corresponding thereto as a marker. Therefore, simply measuring the presence of each marker classified into two groups can predict the postoperative prognosis or metastatic potential of cancer patients in vitro.

Biological specimens include cancer tissues or cells that have been removed by surgery, tissues or cells obtained by biopsy, and the like.
Hereinafter, these two different methods will be described in detail.

3.1 Method Using Polynucleotides In order to detect the 45 genetic markers involved in the present invention, the above polynucleotides that hybridize with each of those markers are used. The number of genes to be detected is 1 or 2 or more per group, preferably 2 or more, more preferably 3 to the total number, for example, 4 to the total number, 5 or more to the total number. The greater the number of genes to be detected, the better the prediction accuracy.

  Hybridization can be performed by a microarray method, a blotting method such as Northern or Southern blotting, Northern or Southern hybridization method, in situ hybridization method, quantitative RT-PCR method or the like. Preferred hybridization methods are microarray, quantitative RT-PCR or blotting. Examples of preferred microarrays are DNA microarrays and protein microarrays.

  In the DNA microarray method, a DNA microarray in which nucleic acid probes that hybridize with each of the above two groups of gene groups (1 to all) are bound to a substrate is used.

  As the DNA microarray, any type of substrate can be used as long as the nucleic acid probe can be immobilized. The solid phase includes, for example, glass, a polymer, and a spacer or a crosslinker including a reactive group for covalently binding a nucleic acid can be introduced. Since such chips are commercially available, it is desirable to use them.

  The solid phase immobilization of the nucleic acid probe is not particularly limited, but a general method such as a method of spotting DNA using a high-density dispenser called a spotter or an arrayer, an ink jet method of ejecting droplets from a nozzle, or the like This method can be used.

  A nucleic acid obtained by labeling nucleic acid such as DNA or RNA in a biological specimen, cDNA or cRNA derived therefrom with a fluorescent substance such as Cy dye (Cr3 or Cy5) is hybridized with a probe on a DNA microarray. The fluorescence intensity is read using a reading device by laser scanning, and the data is analyzed by a computer.

In the blotting method, the nucleic acid probe of the present invention is labeled with a radioisotope (for example, ³² P and ³⁵ S) or a fluorescent substance (rhodamine derivative, Cy dye, etc.) and then transferred to a polymer membrane such as nylon. Hybridization is performed with DNA or RNA in the sample, and nucleic acids such as cDNA and cRNA derived therefrom. The signal is detected using a radiation detector or a fluorescence detector and its intensity is measured.

  In the quantitative RT-PCR method, PCR is performed by annealing primers with cDNA so that target gene regions can be amplified using cDNA prepared from RNA in biological specimens as a template. Is detected. Detect and quantify target genes by pre-labeling primers with radioisotopes or fluorescent materials, or by electrophoresis of PCR products on agarose gel and staining double-stranded DNA with ethidium bromide can do.

PCR conditions are, for example, denaturation: 92 to 94 ° C. for 30 to 60 seconds; annealing: 50 to 55 ° C. for 30 to 60 seconds; extension: 68 to 72 ° C. for 30 to 60 seconds, and 30 to 40 cycles of reaction. including. Reverse transcriptase, commercially available enzymes, for example SuperScript (TM) III (Invitrogen, USA), AMV Reverse Transcriptase (Promega, ^{USA), M-MLV (RNaseH -} ) ( Takara Shuzo, Kyoto) can be used such as .

  In the present invention, the hybridization may be any of DNA-DNA hybridization, DNA-RNA hybridization, and RNA-RNA hybridization.

  Hybridization is usually performed under stringent conditions. Such conditions include, for example, hybridization in 1 M sodium chloride / 0.5% (W / V) sarkosyl / 30% formamide at 60 ° C. for 17 hours, followed by 6 × SSC / 0.005% (W / V V) Triton X-102 solution, once at room temperature for 10 minutes, and further 5 minutes while maintaining at 0-4 ° C. in 0.1 × SSC / 0.005% (W / V) Triton X-102 solution Including one wash. Alternatively, another hybridization condition is, for example, hybridization in 2-6 × SSC at about 45-50 ° C. followed by 0.2-2 × SSC / 0.1 at about 50-65 ° C. Wash with ˜1% SDS; alternatively, 6 × SSC at 60-65 ° C., Denhardt solution, 0.2% SDS after hybridization in 0.2 × SSC, 0.1% at 60-65 ° C. Includes washing with SDS. For conditions and methods of hybridization, see, for example, Ausubel et al., Current Protocols in Molecular Biology, 1989, John Wiley and Sons, US).

3.2 Method by antibody An alternative method for measuring the expression level of the gene is an immunological method.
Antibodies produced as described above can be used for the detection of target polypeptides or fragments thereof in biological specimens.

  Detect or quantify multiple target polypeptides at once by creating a protein microarray with multiple antibodies bound on a microarray substrate, or by spotting multiple antibodies in a dot-like pattern on a PVDF membrane or other filter It becomes possible to do. Or, conventional immunological measurement methods such as enzyme immunoassay (ELISA, EIA), fluorescent antibody method, radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetric method, latex agglutination, latex turbidimetric The target polypeptide or fragment thereof in a biological sample can be detected or quantified by a method, hemagglutination reaction, particle agglutination reaction, Western blotting, or the like.

  When the reaction is performed on a solid phase, solid phase carriers include films (filters) of polymers such as polystyrene, polycarbonate, and polyethylene, plates, tubes, strips, and particles such as latex and magnetic materials. The solid phase can be physically or chemically performed. For chemical coupling, the solid phase can be treated with a reagent such as a maleating reagent or cyanogen bromide to introduce a functional group that reacts with the amino group of the protein into the solid phase.

For detection of the target, the antibody may be labeled, or a labeled secondary antibody may be used. Labels include enzymes such as horseradish peroxidase and alkaline phosphatase, fluorescent materials such as fluorescein, rhodamine and derivatives thereof, luminescent materials such as luciferase and luminol, and radioactive isotopes such as ³² P, ¹²⁵ I and ³⁵ S Is included. Labeling includes, for example, glutaraldehyde method, maleimide method, pyridyl disulfide method, chloramine T method, Bolton Hunter method and the like.

4). Screening for Cancer Metastasis Inhibitor The present invention further uses cultured cancer cells to inhibit or suppress a cancer metastasis-related gene comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 27 or a transcription product thereof, or of cultured cancer cells. Provided is a method for screening a cancer metastasis inhibitor comprising screening a candidate drug for inhibition or suppression of motility and / or invasive ability.

  Specifically, this method comprises preparing cultured cancer cells, culturing the cells in the presence of a candidate drug, and inhibiting or suppressing the expression of the cancer metastasis-related gene or transcription product thereof, or of cultured cancer cells. Screening candidate drugs for inhibition or suppression of motility and / or invasive ability.

  The cell line of human cultured cancer cells, preferably human cultured metastatic cancer cells, is not particularly limited by the type of cancer. For example, a well-known metastatic human cancer cell line, MeWo; malignant melanoma cell line, MDA -MB-435; breast cancer cell line, LNCaP or PC-3; highly metastatic human lung cancer cell line, LNM35; prostate cancer cell line and the like, which can be used in the present invention.

  In the present invention, the degree of inhibition or suppression of the expression of the human cancer metastasis-related gene or its transcription product can be determined by a comparison experiment with a control to which no candidate drug is added. Expression levels can be determined for total RNA or mRNA or poly A (+) RNA obtained from cancer cell lines by well-known methods (eg, phenol / chloroform extraction and ethanol precipitation, oligo dT cellulose column chromatography, etc.), or reverse transcriptase − CDNA synthesized from RNA by PCR (RT-PCR) can be determined by a hybridization method using a fluorescent or radiolabeled probe (eg, Northern hybridization, Southern hybridization, DNA microarray, tissue microarray, etc.) . Alternatively, the expression level is indirectly measured by measuring the intracellular level of the polypeptide encoded by the human cancer metastasis-related gene by an immunoassay, Western hybridization or the like using an antibody against the polypeptide or a fragment thereof. Can be determined.

  The probe is a nucleotide sequence of SEQ ID NO: 1-45 or a sequence complementary thereto, or a sequence thereof, for example, about 20 or more, about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, 250 or more. DNA having a sequence consisting of nucleotides. The probe is preferably a labeled probe bound with a fluorescent or radioactive label. Fluorescent labels include, for example, fluoresamine, rhodamine, derivatives thereof, Cy3, Cy5, etc. Radiolabels include, for example, radioactive phosphorus or sulfur atoms.

  The immunoassay is an analysis method using an antigen-antibody reaction, and can be performed by a method that appropriately combines, for example, an enzyme-linked antibody method (for example, ELISA), a fluorescent antibody method, a solid phase method, a homogeneous method, and a sandwich method. it can. These methods are well known in the art and their conventional techniques can be used in the present invention.

  In human cultured (metastatic) cancer cells, when the expression of the human cancer metastasis-related gene or the corresponding mRNA is significantly inhibited or suppressed by the presence of the candidate drug as compared to the control without the candidate drug, Candidate agents can be identified as cancer metastasis inhibitors.

  Candidate drug screening can also be found by examining inhibition or suppression of motility and / or invasive potential of human cultured cancer cells, preferably metastatic cancer cells.

  The in vitro motility assay or invasive ability assay of cancer cells can be performed, for example, using a transwell chamber culture system. In the motility assay, for example, an insert (Becton Dickinson) having a polyethylene terephthalate film having a small pore having a diameter of 8 microns is inserted into a 24-well cell culture plate, and metastatic cancer cells are placed in a serum-free cell medium inside the insert. This can be done by inoculating the strain and culturing for 24 hours, and then counting the number of cells that have passed through the stoma and migrated to the lower membrane (Kozaki K et al., Cancer Research 60: 2535-40, 2000). The invasive capacity assay uses a similar system, but can be measured by covering the top of the membrane with Matrigel before seeding the cells (Kozaki K et al., Cancer Research 60: 2535-40, 2000). .

  In the above assay, by adding a candidate drug to the serum-free cell medium, a candidate substance that inhibits or suppresses the motility and / or invasive ability of metastatic cancer cells can be identified as a cancer metastasis inhibitor.

  Candidate agents include, but are not limited to, small molecules, peptides, polypeptides, proteins, nucleosides, oligonucleotides, polynucleotides, nucleic acids (DNA or RNA), and the like.

  Examples of the polypeptide or protein as a candidate drug include an antibody or a fragment thereof against the polypeptide or protein encoded by the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof. The nucleic acid includes a ribozyme capable of cleaving mRNA corresponding to the base sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, siRNA (small interfering RNA) and the like.

  In order to select the sequence of the siRNA, for example, known knowledge for selection of the target site of the target mRNA, eg (a) the GC content is about 30 to about 70%, preferably about 50%, (b) Criteria can be used such that all nucleotides are equal and G is not contiguous, (c) the 5 ′ terminal nucleotide of the antisense strand is A, U, etc. (DM Dykxhoorn) Et al., Nature Rev. Mol. Cell Biol. (2003), 77: 7174-7181; A. Khlovova et al., Cell (2003), 115: 209-216).

The ribozyme as a candidate drug is RNA having catalytic activity and has an activity of cleaving mRNA corresponding to the target human cancer metastasis-related gene according to the present invention. This cleavage inhibits or suppresses the expression of the gene. It is known that a ribozyme-cleavable target sequence is generally a sequence containing NUX (N = A, G, C, U; X = A, C, U), for example, a GUC triplet. Since the candidate triplet is present in the corresponding mRNA sequence encoded by the nucleotide sequence of SEQ ID NO: 1-27 of the target human cancer metastasis-related gene in the present invention, the mRNA sequence portion to be cleaved including the candidate triplet Ribozymes containing sequences complementary to can be used for cleavage of the target mRNA. Such ribozymes include hammerhead ribozymes. The hammerhead ribozyme is composed of a nucleotide sequence that constitutes a sensor site, a nucleotide sequence that includes a region that can form a cavity that stably captures Mg ²⁺ ions only when RNA is bound to the sensor site, and a region around the target RNA cleavage site. Nucleotide sequences that include regions that are complementary to the sequence can be included.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

(Establishment of highly metastatic human lung cancer cell line (LNM35))
NCI-H460 cells (obtained from American Type Culture Collection) (parent strain) 1 × 10 ⁷ cells / 100 μl (RPMI 1640 cell culture medium) were subcutaneously transplanted into SCID mice (purchased from Shizuoka Laboratory Animal Research Institute). 45 days after transplantation, lung metastasis of cancer cells was confirmed, and cancer cells from lung metastases were transplanted subcutaneously into another mouse, and cancer cells from lung metastases were cultured in culture medium (10% bovine serum). Primary culture was performed in supplemented RPMI 1640). The cells were transplanted subcutaneously into another mouse, and similarly cancer cells from lung metastases were cultured. Furthermore, cells after two in vivo selections were transplanted subcutaneously into mice, and cancer cells from axillary lymph nodes were subjected to primary culture in a culture medium (RPMI 1640 supplemented with 10% bovine serum). Subsequently, cloning was performed by a limiting dilution method using a 96-well plate to obtain a highly metastatic strain NCI-H460-LNM35 (FIG. 1).

  Separately, a low metastatic strain (N15) could be obtained from the parent strain NCI-H460 cells by limiting dilution. Furthermore, by treating the LNM35 strain with a demethylating agent (5-aza-2′-deoxycytidine) (1 μM for 24 hours) in the same culture medium as described above, low metastases (L2D2 and L2D3A) were treated. I was able to get it.

  The LNM35 strain and the parent strain obtained by the above procedure were examined for their ability to metastasize by cell motility assay and invasive ability assay, and the ability of metastasis to axillary lymph nodes and lungs by subcutaneous transplantation into mice.

  The motility assay was carried out using a filter transwell chamber with 8 μm micropores and a procedure for counting the number of cells that had migrated to the back of the filter after 24 hours of culture.

The invasion ability assay was performed by using a transwell chamber in which a filter having 8 μm micropores was coated with Matrigel and counting the number of cells migrated to the back side of the filter after 24 hours of culture.
Lymph node and lung metastases were determined by weight and number of masses, respectively.

  As a result, as shown in FIG. 2, the LNM35 strain was the most highly metastatic cancer cell line because it showed the highest levels of both motility and invasive potential, and also showed high levels of lymph node and lung metastasis. It was confirmed. On the other hand, all of the parent strains showed very low motility, invasive ability, lymph node metastasis, and lung metastasis, confirming that they were low metastasis cancer cell lines.

(Detection of novel cancer metastasis-related genes)
Using GeneFilter Human Microarrays Release I and Release II (registered trademark) microarray (manufactured by Invitrogen), detection of a specific gene exhibiting a gene expression difference between the highly metastatic strain LNM35 and the parent strain N15 was examined.

Specifically, in this microarray, 5 μg of total RNA was used as a template, oligo-dT primer and SuperScript II reverse transcriptase were used to perform reverse transcription in the presence of 100 μCi of [ ³³ P] dCTP, and a cDNA microarray (GeneFilter manufactured by Invitrogen) was used. Hybridized with Human Microarrays Release I and Release II). 2M of urea, 0.1% of SDS, 50 mM of Naphosphate buffer (pH 7.0), 150 mM of NaCl, 1 mM of MgCl ₂ and 0.2% AlkPhos DIRECT blocking reagent (manufactured by Amersham) After that, the microarray was brought into contact with the imaging plate (Fuji Photo Film) for 2 hours, and the imaging plate was scanned using BAS5000 (Fuji Photo Film), so that all 11 corresponding to 8,644 genes were obtained. , 168 spot expression profiles were obtained twice.

(Bioinformatics analysis)
In the expression profiles of LNM35 and N15 twice, a total of 4 times, spots with an expression level (scanned spot value) of 0.1 or less were excluded from analysis due to detection limit.

  After normalizing the expression profile four times using the lowess method which is a non-linear correction method, the average value was calculated | required about each of LNM35 and N15.

  The expression profiles of LNM35 and N15 were compared, and spots with an expression difference of 2SD (2 times the standard deviation) or more were extracted.

  Furthermore, in the spot where the expression is enhanced in LNM35 for the extracted spot, both the expression values of the two expression profiles of LNM35 are greater than 1.0, and in the spot where the expression is enhanced at N15, When spots were searched that matched the condition that the expression values of the two expression profiles of N15 were both greater than 1.0, spots corresponding to 45 genes were extracted.

(Patient case)
Fifty lung cancer patient cases were obtained from a file of successful patients who underwent curative resection at the Aichi Cancer Center Thoracic Surgery (Nagoya). All tumor specimens were embedded in OCT compounds and stored at −80 ° C. after obtaining the required approval from the formal review department and written informed consent from the patient.

(Acquisition of expression profile)
About 50 sections were cut from the frozen tissue of the tumor specimen to a thickness of about 7 μm. Every 10 sections were stained with Giemsa, and under the guidance of a pathologist, total RNA was extracted from the region where most of the tumor tissue occupies using an RNeasy kit (Qiagen, USA). About 5 μg of total RNA was reverse transcribed in the presence of 100 μCi [ ³³ P] dCTP using oligo-dT primer and SuperScript II reverse transcriptase. After hybridization with Invitrogen cDNA microarray (GeneFilter Human Microarrays Release I and Release II), the microarray was contacted with an imaging plate (Fuji Film) for 2 hours, and imaged using BAS5000 (Fuji Film). By scanning the plate, an expression profile of all 11,168 spots corresponding to 8,644 genes was obtained twice.

(Bioinformatics analysis)
After correcting the expression profile obtained using the rank-invariant method, the expression value was determined from the average value of two times.

(Relationship between expression patterns of 45 metastasis-related genes and recurrence / death)
Based on the similarity of 45 genes extracted from the comparison of LNM35 and N15, cluster analysis was performed on data of 50 lung cancer patients who underwent lung cancer surgery at the Aichi Cancer Center (FIG. 3A).

  The dots shown in red or green in the figure indicate the expression state of each gene in each case. The red color indicates that the gene expression is relatively high compared to other cases, and the green color indicates that the gene expression is relatively low. Further, the tree diagrams in the figure show similarity. If the similarity is high, the branches indicating the connection of the tree diagrams are short, and if the similarity is low, the branches indicating the connection of the tree diagrams are long. The cases are linked in descending order of the degree of similarity, and are finally compiled as one tree diagram.

  When 50 cases of lung cancer specimens were subjected to clustering analysis, they were classified into two large groups. In the left group, most of the genes with high expression (red dots) were genes whose expression was enhanced by LNM35. On the other hand, in the right group, most of the genes with high expression were genes whose expression was suppressed by LNM35. Here, when one group on the left side was a Fatal group and one group on the right side was a Favorite group, Kaplan-Meier survival analysis was performed on the prognosis of these two groups, and a prognostic difference was detected with a significant difference (P = 0.0234). (FIG. 3B). As a result, the prognosis of the group in which the expression of the gene group whose expression is enhanced in LNM35 is relatively high (Fatal group) is poor, and the group in which the expression of the gene group whose expression is suppressed in LNM35 is relatively high (Favorable group). We were able to confirm that the prognosis of the group was good using data on human lung cancer cases.

  FIG. 4A shows the result of cluster analysis using data of 62 lung cancer patients acquired at Harvard University.

  We analyzed the data obtained at other facilities and examined whether the same results as in Fig. 3 could be obtained. Because Harvard's data is different from our data acquisition method, only expression information on 38 genes out of 45 genes could be obtained. Using the expression information of these 38 genes, the same analysis as in FIG. 3 was performed.

  62 cases of lung cancer specimens were subjected to clustering analysis and classified into two large groups. In the group on the right side, most of the genes with high expression (red dots) were genes whose expression was enhanced by LNM35. On the other hand, in the left group, most of the highly expressed genes were genes whose expression was suppressed by LNM35. Here, when one group on the right side is a Fatal group and one group on the left side is a Favorite group, Kaplan-Meier survival rate analysis was performed on the prognosis of these two groups, and a prognostic difference was detected with a significant difference (P = 0.0385). (FIG. 4B). As a result, the prognosis of the group in which the expression of the gene group whose expression is enhanced in LNM35 is relatively high (Fatal group) is poor, and the group in which the expression of the gene group whose expression is suppressed in LNM35 is relatively high (Favorable group). The prognosis of group) was good, even using data from human lung cancer cases acquired at Harvard University.

  FIG. 5A shows the result of cluster analysis using the data of 79 breast cancer patients acquired at the National Cancer Institute of the Netherlands.

  The data obtained at other institutions were analyzed, and it was examined whether the same results as in FIG. 3 could be obtained for solid cancers other than lung cancer. Since the data from the Dutch National Cancer Institute is different from our data acquisition method, we were able to obtain only expression information for 37 genes out of 45 genes. Using the expression information of these 37 genes, the same analysis as in FIG. 3 was performed.

  79 breast cancer specimens were subjected to clustering analysis and classified into two large groups. In the left group, most of the genes with high expression (red dots) were genes whose expression was enhanced by LNM35. On the other hand, in the right group, most of the genes with high expression were genes whose expression was suppressed by LNM35. Here, when one group on the left side is a Fatal group and one group on the right side is a Favorite group, Kaplan-Meier survival rate analysis was performed on the prognosis of these two groups. As a result, a prognostic difference was detected with a significant difference (P = 0.0032). (FIG. 5B). As a result, the prognosis of the group in which the expression of the gene group whose expression is enhanced in LNM35 is relatively high (Fatal group) is poor, and the group in which the expression of the gene group whose expression is suppressed in LNM35 is relatively high (Favorable group). The prognosis of the group) was good, even using data from human breast cancer cases obtained at the Dutch National Cancer Institute.

(Multivariate analysis by Cox proportional hazard model)
The prognostic index by 45 genes extracted from the comparison of LNM35 and N15 was examined (FIG. 6).

  Whether the 45-gene postoperative prognostic index is effective as a postoperative prognostic factor, age (over 63 years / under 63 years), gender (male / female), smoking history, using the proportional hazard model of COX Examination was performed by multivariate analysis using six items: (no smoking history / present), tissue type (squamous cell carcinoma / non-squamous cell carcinoma), stage (pSTAGE), and metastasis signature (Favorable / Fatal). As a result, it was shown with statistical significance that only two items of stage and metastasis signature by 45 genes are effective as postoperative prognostic factors (stage: P = 0.014, depending on 45 genes) Metastasis signature: P = 0.039). In the figure, hazard ratio indicates a hazard ratio, 95% CI indicates a 95% confidence interval, and P value indicates a risk rate.

(Example of identifying cancer with good or bad postoperative prognosis)
A signal-noise function (signal-to-noise metrics, Golub et al., Science, Vol. 286, pp531 to 537, 1999) was used to classify cancers with good or bad postoperative prognosis. When the cancer case group is defined as class 1 for cancer with a good postoperative prognosis and class 2 for cancer with a poor postoperative prognosis, a weight S that is a signal-noise statistical value is calculated by the following equation.
S = (μ _class1 −μ _class2 / σ _class1 + σ _class2 )

Here, for each gene, mu _class1 represents the average of data on total expression strength of class1, mu _class2 represents the average of data on total expression strength of class2, sigma _class1 is the standard deviation of data on total expression strength of class1 Σ _class2 indicates the standard deviation of all expression intensity data of class2.

Next, a weighted vote for gene x is calculated using the following weighted-voting formula.
_{V x = S (G x -b} x)

Here, V _X represents a weighted vote for gene x, S represents a weight calculated by the above equation, G _X represents the expression intensity (or expression level) of gene x, and b _X is
b _X = (μ1 + μ2) / 2
(Where μ1 and μ2 indicate the average of the average values of class1 and class2, respectively) and indicate the centers (ie, centroids) of the two groups.

The technique will by adding the weights in accordance with a deviation from the center of gravity, when the sum is greater than 0 V _X of each group, cancers are classified into class1, when the sum of V _X is smaller than 0, cancer in class2 Can be classified.

For example, for samples 1, 2, 3, 4 and 5 belonging to class 1 and samples 6, 7, 8, 9 and 10 belonging to class 2, genes (Gene) A, B, and C (characterized as having good postoperative prognosis) Expression intensity (or expression level) of genes (Gene) X, Y, Z (genes characterized as having poor postoperative prognosis), and expression of each gene in five samples of each group The average value (μ) and standard deviation (σ) of the intensity are calculated, and further, the weight (S) and the center of gravity (b _X ) are calculated from the above formula.

For sample A and sample B to be typed, the expression intensity (or expression level) of each gene is measured, and the weight (S) and G _x -b _x are calculated from the above formula to obtain each V _X. Find the sum of V _X of two genes.

As a result, sample A, because the sum of _{V X} is greater than 0, it is identified as class1. Moreover, sample B, because the sum of _{V X} is less than 0, is identified as class2.

  According to the present invention, the possibility of cancer metastasis and the prognosis of cancer patients can be predicted, which can greatly contribute to cancer treatment planning and prognosis management.

The establishment procedure of human highly metastatic lung cancer cell line LNM35 is shown. The comparison of the motility and invasion ability in cultured cells and the ratio of lymph node metastasis and lung metastasis after mouse transplantation between LNM35 strain and its parent strain is shown. When applied to 50 cases of lung cancer data from the Aichi Cancer Center, lung cancer patients are roughly divided into two groups based on the correlation between the expression pattern of 45 metastasis-related genes and the proportion of survivors after surgery. Indicates. FIG. 3A shows the result of hierarchical clustering analysis. FIG. 3B shows the results of Kaplan Meier survival analysis (horizontal axis: number of months). When applied to 62 cases of lung cancer from Harvard University (Bhattacharjee A et al., Proc Natl Acad Sci USA 98: 13790-95, 2001), lung cancer patients were found to have 45 metastasis-related gene expression patterns and postoperative survivors. The correlation with the ratio is roughly divided into two groups. FIG. 4A shows the result of hierarchical clustering analysis. FIG. 4B shows the results of Kaplan Meier survival analysis (horizontal axis: number of months). When applied to 79 breast cancer data from the National Cancer Institute (van't Veer LJ et al., Nature 415: 530-6, 2002), breast cancer patients had 45 metastasis-related gene expression patterns and postoperative survivors It is shown that the correlation with the ratio is roughly divided into two groups. FIG. 5A shows the result of hierarchical clustering analysis. FIG. 5B shows the results of Kaplan Meier survival analysis (horizontal axis: number of months). The risk factors for 5-year survival after surgery are shown as predictors of recurrence and death independent of the stage determined by multivariate analysis using the Cox proportional hazard model.

Claims (18)

  A method for predicting postoperative prognosis or metastasis potential in cancer patients in vitro, wherein the cancer metastasis comprises a nucleotide sequence shown in SEQ ID NOs: 1 to 45 or a mutant sequence thereof in a biological specimen derived from the patient The level of expression of at least one related gene marker or its transcriptional or translational product level is measured using a probe corresponding to the marker, and the postoperative prognosis is better or the patient is less likely to metastasize. The method, comprising determining postoperative prognosis or metastatic potential using the relative difference in level between patients with a worse prognosis or a higher probability of metastasis as an index.   When the cancer metastasis-related gene marker consists of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient is identified as a patient with a better prognosis after surgery or a lower possibility of metastasis. The method of claim 1.   When the cancer metastasis-related gene marker consists of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, the patient is identified as a patient with a worse prognosis after surgery or a higher possibility of metastasis. The method of claim 1.   The probe comprises a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1-45, a complementary polynucleotide thereto, a variant thereof, a polynucleotide that hybridizes to them under stringent conditions, or 15 or more consecutive sequences 4. The method according to any one of claims 1 to 3, wherein the method is selected from the group consisting of those fragments containing a selected base.   The probe is an antibody against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof, or a fragment thereof. The method of any one of 1-3.   The method according to claim 5, wherein the antibody is a polyclonal antibody, a monoclonal antibody, or an anti-peptide antibody.   The method of any one of claims 1 to 6, wherein the cancer is a solid cancer.   The method according to claim 7, wherein the solid cancer is lung cancer or breast cancer.   A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1-45, a polynucleotide complementary thereto, a variant thereof, a polynucleotide that hybridizes to them under stringent conditions, or contains 15 or more consecutive bases A composition for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, comprising at least one probe selected from the group consisting of those fragments.   A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 28 to 45, a polynucleotide complementary thereto, a variant thereof, a polynucleotide that hybridizes to them under stringent conditions, or 15 or more consecutive bases A composition for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, comprising at least one probe selected from the group consisting of those fragments.   A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 27, a polynucleotide complementary thereto, a variant thereof, a polynucleotide that hybridizes to them under stringent conditions, or 15 or more consecutive bases A composition for predicting in vitro the postoperative prognosis or metastatic potential of cancer patients, comprising at least one probe selected from the group consisting of those fragments.   At least one selected from the group consisting of an antibody against a polypeptide selected from the group consisting of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof. A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising two probes.   At least one selected from the group consisting of an antibody against a polypeptide selected from the group consisting of a polypeptide consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a polypeptide encoded by a variant thereof, or a fragment thereof. A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising two probes.   At least one selected from the group consisting of an antibody against a polypeptide selected from the group consisting of a polypeptide consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a polypeptide encoded by a variant thereof, or a fragment thereof. A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising two probes.   15. A composition according to any one of claims 9 to 14, wherein the probe is included in the form of a kit.   The composition according to any one of claims 9 to 11, wherein the probe is included in the form of a microarray.   Using cultured cancer cells, inhibition or suppression of a cancer metastasis-related gene comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 27 or a transcription product thereof, or inhibition or suppression of motility and / or invasive ability of cultured cancer cells A screening method for a cancer metastasis inhibitor, comprising screening a candidate drug.   Preparing cultured cancer cells and culturing the cells in the presence of a candidate drug to inhibit or suppress the expression of the cancer metastasis-related gene or its transcription product, or to inhibit the motility and / or invasive ability of the cultured cancer cells Or the method of claim 17 comprising screening candidate agents for inhibition.

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