WO2007077977A1 - Composition and method for predicting the postoperative prognosis or metastatic risk of cancer patient - Google Patents

Abstract

A method of predicting in vitro the postoperative prognosis or metastatic risk of a cancer patient which comprises measuring the expression level of at least one cancer metastasis-related gene marker in a biological sample originating in the patient or the transcription or translation product level thereof by using a probe corresponding to the marker, and then determining the postoperative prognosis or metastatic risk with the use of a difference in the relative level as discussed above between one group of patients showing a better postoperative prognosis or a lower metastatic risk and another group of patients showing a worse postoperative prognosis or a higher metastatic risk as an indication; and a composition for predicting the postoperative prognosis or metastatic risk which contains the probe as described above.

Description

 Composition and method for predicting postoperative prognosis or metastatic potential of cancer patients

Technical field

 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 provides a method for screening a cancer metastasis inhibitor using cultured cancer cells.

Regarding the method. book

Background art

 Cancer is a multifactorial disease in which genes and environmental factors are complicatedly involved. Various therapies such as chemotherapy, radiation therapy, surgery, and immunotherapy have been implemented, but due to the complexity of cancer onset mechanisms, it is imperative to treat mid-stage and end-stage cancer and to suppress cancer metastasis. However, it cannot be said that sufficient effects have been achieved.

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

 One of the major causes that hinders cancer treatment and recurrence is cancer metastasis. Because of the motility and invasive ability of cancer, cancer spreads remotely from the primary focus to another tissue and grows. The overall picture of this transfer mechanism is largely unknown. As cancers become more malignant, and when cancers are removed, cancers are more likely to metastasize, resulting in recurrence of the cancer, so preventing cancer migration is an early diagnosis and effective treatment method for cancer. Along with development, it has become an important issue in the medical field.

Currently, several cancer metastasis-related genes have been reported, most of which have been discovered by the differential display method. This method is to detect the target gene based on the difference in the present pattern. Many of the genes found by this method are genes that are expressed in a certain amount in common in many tissues and cells. , This is a so-called housekeeping 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 the human VEGF receptor F 1 t _ l (Patent Document 1), a peptide having cell adhesion inhibitory activity (Patent Document 2), a function of purine receptor There are known purine compounds (Patent Document 3) and the like. 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 method, ribozyme, RNA interference (RNA i) and the like (Non-patent Document 1). In particular, RNA i is highly specific, so basic research for its medical application is rapidly progressing, and its target is viral disease, cancer, genetic disease, etc. Patent Document 2).

 Patent Document 1 Japanese Patent Laid-Open No. 2 003- 26 1 46 0

 Patent Document 2 Japanese Patent Application Laid-Open No. 09-143076

 Patent Document 3 Japanese Unexamined Patent Publication No. 7-08 2 1 56

 Patent Document 4 Japanese Table 9 8Z04 543 1

 Non-Patent Literature 1 Daisuke Seki et al., Experimental Medicine 2 2-14 14 (extra number), 89-98 pages, 2 004, Yodosha (Tokyo, Japan)

 Non-Patent Literature 2 Experimental Medicine 2 2-4 “RN A i Science”, 2004, Yodosha (Tokyo, Japan) Disclosure of Invention

So far, it has been suggested that certain genes or polypeptides may be associated with cancer metastasis, but at the laboratory level they are associated with metastasis, but other systems may The association could not be confirmed, or it was sufficient to develop an inhibitor and apply it clinically There were many events that were not effective.

 In such a situation, the present inventors have focused on the fact that metastasis is not caused by a single gene but by a coordinated interaction of a plurality of gene groups, identifying cancer metastasis-related gene markers, and The relationship between the gene marker 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 metastatic potential of cancer patients in vitro using a cancer metastasis-related gene group as a marker. Another object of the present invention is to provide a method for screening a cancer metastasis inhibitor based on inhibition or suppression of a cancer metastasis-related gene or a transcription product thereof. Comprehensive gene expression analysis was performed between the highly metastatic human lung cancer cell line developed by the present inventors and its low metastatic parent strain, and the difference in expression was shown in both questions that are considered to be related to the acquisition of metastasis. We identified genes and examined 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 multiple carcinomas, the cancer patient group is roughly divided into two groups according to the expression profile of the identified gene group, and the survival period (or prognosis) after surgery is divided into the two groups. It was found that there was a significant difference.

Summary of the Invention

 In summary, the present invention has the following characteristics.

 (1) A method for predicting postoperative prognosis or metastasis potential in cancer patients in vitro, the base sequence shown in SEQ ID NOs: 1 to 45 or a mutant sequence thereof in a biological sample derived from the patient At least one expression level or transcription or translation product level of a cancer metastasis-related gene marker containing a marker is measured using a probe corresponding to the marker, so that the prognosis after surgery is better or the possibility of metastasis is increased. To determine the postoperative prognosis or metastatic potential using the relative difference in level between the lower patient group and the patient group with worse postoperative prognosis or higher metastatic potential. Including the above method.

(2) When the cancer metastasis-related gene marker is composed of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient has a better postoperative prognosis or is less likely to transfer. The method according to (1) above, which is identified as a patient. (3) When the cancer metastasis-related gene marker comprises the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, the patient is a patient with a worse prognosis after surgery or a higher possibility of metastasis. The method described in (1) above, identified as

 (4) A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45, a polynucleotide complementary thereto, a variant thereof, and a polynucleotide that hybridizes to them under stringent conditions Or the method according to any one of (1) to (3) above, which is selected from the group consisting of those fragments containing 15 or more consecutive bases.

 (5) The probe is 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, or the The method according to any one of (1) to (3) above, which is a fragment.

 (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 to 4, a complementary polynucleotide, a variant thereof, a polynucleotide that hybridizes under stringent conditions thereto, or 15 or more A composition for predicting postoperative prognosis or metastasis potential in cancer patients in vitro, comprising at least one probe selected from the group consisting of those fragments comprising a sequence of nucleotides.

 (10) Polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 28 to 45, polynucleotide complementary thereto, variant thereof, polynucleotide hybridizing under stringent conditions, or 15 or more A composition for predicting in vitro the postoperative prognosis or metastasis potential of cancer patients, comprising at least one probe selected from the group consisting of those fragments comprising a continuous base of.

(1 1) Polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 27, polynucleotide complementary thereto, variant thereof, polynucleotide hybridizing under stringent conditions, or 1 5 or more consecutive bases A composition for predicting postoperative prognosis or metastatic potential of cancer patients after surgery, comprising at least one probe selected from the group consisting of those fragments.

 (1 2) A group consisting of 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. A composition for predicting in vitro the postoperative prognosis or metastasis potential of cancer patients, comprising at least one probe selected from:

 (1 3) A group consisting of 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: 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 at least one probe selected from:

 (14) consisting of 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 A composition for predicting in vitro the postoperative prognosis or metastatic potential of a cancer patient, comprising at least one probe selected from the group.

 (15) The composition according to any one of (9) to (14), wherein the probe is included in the form of a kite.

 (16) The composition according to any one of the above (9) to (11), wherein the probe is included in the form of a microarray.

 (1 7) Inhibition or suppression of cancer metastasis-related genes or transcripts thereof containing any of the nucleotide sequences of SEQ ID NOS: 1 to 27 using cultured cancer cells, and the motility of Z or human cultured cancer cells And / or a screening method for a cancer metastasis inhibitor, comprising screening a candidate drug for inhibition or suppression of invasive ability.

(18) Preparing cultured cancer cells, culturing the cells in the presence of a candidate drug, and inhibiting or suppressing the expression of the cancer transfer-related gene or its transcription product, and the motility of Z or cultured cancer cells And the method of (17) above, comprising screening a candidate drug for inhibition or suppression of Z or invasive 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 metastasis potential of cancer patients in multiple carcinomas. I found out. By using these markers as indicators, it is possible to predict the postoperative prognosis of cancer patients and the possibility of cancer metastasis, so it is possible to formulate treatment plans for patients, improve treatment outcomes for cancer, and recurrence due to metastatic cancer. It can be expected to be of great use for prevention and prognosis management. Furthermore, in cultured cancer cells, it is possible to screen for a drug that regulates its expression using the above marker as an index, which leads to the development of a drug that suppresses or prevents cancer metastasis. it can.

 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2005-379867, which is the basis of the priority of the present application. Brief Description of Drawings

 Fig. 1 shows the procedure for establishing the human highly metastatic lung cancer cell line LNM35.

 FIG. 2 shows a comparison of the motility and invasion ability (A) in cultured cells and the ratio of lymph node metastasis and lung metastasis (B) after mouse transplantation between the LNM3 5 strain and its parent strain. Figure 3 shows the correlation between the expression pattern of 45 metastasis-related genes and the proportion of survivors after surgery when applied to 50 cases of lung cancer data from the Aichi Cancer Center (Nagoya, Japan). Shown to be roughly divided into two groups. Figure 3A shows the results of hierarchical clustering analysis. Figure 3B shows the results of force planmeyer survival analysis (vertical axis: survival rate, horizontal axis: number of months).

Figure 4 shows that when applied to Harvard University (USA) 62 cases of lung cancer data (Bhattacharjee A et al., Proc Natl Acad Sci USA 98: 1 3 790-9 5, 200 1) The correlation between the expression pattern of 45 metastasis-related genes and the proportion of survivors after surgery is broadly divided into two groups. Figure 4A shows the results of hierarchical clustering analysis. Figure 4B shows the results of the force plan meyer survival analysis (vertical axis: survival rate, horizontal axis: number of months). Figure 5 is applied to 7 9 cases of breast cancer (van't Veer LJ et al., Nature 4 1 5: 5 30-6, 2002) from the Netherlands National Cancer Institute (Netherlands). We show that breast cancer patients are roughly divided into two groups regarding the correlation between the expression pattern of 45 metastasis-related genes and the proportion of survivors after surgery. Figure 5A shows the results of hierarchical clustering analysis. Fig. 5B shows the results of a force plan myria survival analysis (vertical axis: survival rate, horizontal axis: number of months).

 Figure 6 shows the recurrence and death predictors independent of stage determined by multivariate analysis with a Co x proportional hazards model for the risk of 5-year survival in lung cancer patients. BEST MODE FOR CARRYING OUT THE INVENTION

Definition

 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 highly advanced, while `` good prognosis '' means It means such invasiveness, metastasis or low progression.

 As used herein, “metastasis” refers to a series of processes by which cancer cells move from the primary site to distant tissue by their motility and invasive potential, where they proliferate and form neoplasms. Say. Metastasis is a major obstacle to cancer treatment because it causes cancer recurrence. Cancer cells that have infiltrated blood vessels or lymphatic vessels are known to 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 the preferred mammal being humans.

As used herein, a “biological specimen” refers to a tissue, cell or body fluid taken from a mammal (preferably human), preferably a cancer tissue or cell. In the present invention, cancer tissue or cells derived from a group of patients with a better postoperative prognosis or a lower likelihood of metastasis and a worse prognosis after surgery 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 gene related to the present invention from those derived from different patient groups. For example, lung cancer, breast cancer, colon cancer, prostate cancer, stomach cancer, esophageal cancer, liver cancer, spleen cancer, kidney cancer, uterine / cervical cancer, ovarian cancer, bladder cancer, brain tumor, thyroid gland Examples include, but are not limited to, cancer, lymphoma, testicular cancer, osteosarcoma, skin cancer, melanoma, and blood cancer (especially leukemia).

 As used herein, the “cancer metastasis-related gene marker” refers to a gene that has a difference in expression level between a high metastatic cancer cell line and a low metastatic cancer cell line. It is a marker that has been released 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, a “variant” is a variant that arises from a biological event such as mutation, polymorphism, alternative splicing, the degeneracy of the genetic code, or a homologue between species. Etc. The mutant includes one or more, preferably one or several, 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 substitutions, deletions, additions, insertions, etc., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more identical to the sequence or a partial sequence thereof Mutants consisting of sequences having sex, and the like. Here, “several” usually means an integer of 10 or less. Identity (%) can also be determined using known B LAST and FAS TA programs with or without gaps (SF A ltschul et al., J. Mo 1. B i o. 2 1 5: 403-4 1 0, 1 990). In general, identity (%) can be calculated as a percentage of the number of matched bases over the total number of bases.

 As used herein, “stringent conditions” include, but are not limited to, at least 80%, preferably at least 90%, more preferably at least 95% identity. This means hybridization and washing conditions that allow nucleotide sequences to hybridize to each other. For example, hybridization and washing conditions in microarray analysis are 1M sodium chloride Z0.5%

(W / V) Sarcosyl Z Hybridizer in 30% formamide at 60 ° C for 17 hours And then 6 XSSC / 0.0 0 5% (WZV) Triton X—1 02 solution, once at room temperature for 10 minutes, and 0.1 XSS CZ0. 005% (W / V) Triton X—This is a condition of washing once in 5 minutes while keeping at 0 to 4 ° C in the solution. Here, 1 XSSC is 15 O mM sodium chloride and 15 mM sodium citrate aqueous solution (pH 7.2). For hybridization, see F. M. A usbel et al., Short Protocolsin M o 1 ecu 1 ar B io 1 ogy , 1 995, John Wiley & Sons, Inc. (USA).

Detailed description

 In the following, the present invention will be described in more detail.

 1. Cancer metastasis-related genes

 The human cancer metastasis-related gene marker of the present invention was found as follows. Metastatic lung cancer cell line known as NCI — H 4 60 (American Type C 1 ture Collection; US Viryua)) (parent strain) A highly metastatic cancer cell line, LNM35, was isolated by subculture of cells from and by in vivo selection using mice based on metastatic potential (see Figure 1). On the other hand, cancer cells with even lower metastases can be obtained from NC I —H 4 60 cells by limiting dilution or treatment with a demethylating agent. In the cultured cell or mouse transplantation test, the LNM3 5 strain showed significantly higher levels of motility, invasion ability, lymph node metastasis, and lung metastasis compared to the parent strain or low-metastasis cancer cell line ( (See Figure 2).

By examining the difference in gene expression between the high-metastasis cancer cell line LNM 35 and the parent or low-metastasis cancer cell line by the genefin microarray (Research Genetics) A human cancer metastasis-related gene comprising the nucleotide sequences of SEQ ID NOs: 1 to 45 was found. As a result of data bank search, these genes are registered in Uni Gene or Gen Bank (NC BI, USA) as shown in Table 1, but they are related to cancer metastasis. It is not known about it. Tables 1 and 2 show the sequence numbers of gene markers with high and low expression in the highly metastatic LNM35 strain, Gen B ank ¾ | ¾ "¾" (A ccessionnumber No, U n. I Gene ID and gene name (G ene. S ym bol, Gene name).

table 1

GenBank Accession number UniGene ID Gene symbol

 1 0057 005787 Hs.478481 ALG3 AsparagineHinked glycosylation 3 homolog

 2 — 006825 Hs.74368 CKAP4 Cytoskeleton-associated protein 4

 3 AK_055043 Hs.193235 CPLX2 Complexin 2

 4 NM— 00151 1 Hs.789 CXCL1 Chemokine (C-X-C motif) ligand 1

 5 NMichi 001 344 Hs.82890 DAD1 Defender against ceJI death 1

 6 NM— 1 39072 Hs.234074 DNER Delta-notch— like EGF repeat-containing transmembrane

 7 NM— 001398 Hs.196176 ECH 1 Enoyl Coenzyme A hydratase 1, peroxisomal

 8 NM— 002070 Hs.77269 GNAI2 Guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide

9 NM— 000516 Hs.125898 GNAS GNAS complex locus

 10 NM 1 207312 Hs. 503749 H2-A and PHA Alpha-tubulin isotype H2-alpha

 1 1 NM I 194247 Hs.516539 HNRPA3 Heterogeneous nuclear ribonucleoprotein A3

 12 N _007040 Hs.1 55218 HNRPUL1 Heterogeneous nuclear ribonucleoprotein U— like 1

 13 NM— 007355 Hs.509736 HSPCB Heat shock 90kDa protein 1, beta

 14 NM— 003897 Hs.76095 ER3 Immediate early response 3

 15 N .006838 Hs.444986 METAP2 Methionyl aminopeptidase 2

 16 NM— 020191 Hs.555965 MRPS22 Mitochondrial ribosomal protein S22

 1 7 NM— 002808 Hs.518464 PSMD2 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 2

 18 AF— 095289 Hs.521097 PTTG3 Pituitary tumo ransf ormi ng 3

 19 N J 94298 Hs. 499709 SLC 16A9 Solute carrier family 16 (monocarboxylic acid transporters), member 9

 20 NM— 003220 Hs.51 9880 TFAP2A Transcription factor AP ~ 2 alpha (activating enhancer Dinding protein 2 alpha)

21 N _021 109 Hs.522584 TMSB4X Thymosin, beta 4, XHinked

 22 NM— 006000 Hs.75318 TUBA1 Tubulin, alpha 1 (testis specific)

 23 刚 — 006001 Hs.349695 TUBA2 Tubulin, alpha 2

 24 NMJ 78014 Hs.533059 TUBB Tubulin, beta polypeptide

 25 NM— 003334 Hs.533273 UBE1 Ubiquitin— activating enzyme El

 26 AA 448396 Hs.509585-Transcribed locus, strongly similar to NP_002148.1 chaperonin 10

27 AA— 668189 Hs. 560965 ― Transcribed locus, strongly similar to XP— 4161 57.1 PREDICTED: similar to snRNP-f

Table 2

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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 sputum cancer. One marker reported so far has been specific to a particular cancer, but it was surprising that the marker of the present invention is a common marker for various cancers. For this reason, for example, lung cancer, breast cancer, large intestine cancer, prostate cancer, stomach cancer, esophageal cancer, liver cancer, knee cancer, kidney cancer, uterine body / neck Solid cancers such as head cancer, 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, the cancer metastasis-related gene marker includes not only the gene comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45 but also mutant genes thereof. Usually, such variants include those produced 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 level or protein level as a transcript or translation product. In the case of a transcript, it is detected as mRNA, cRNA or cDNA. Extract total RNA from cells or tissues using extraction methods such as phenolic mouth form Z-soamyl alcohol, guanidinium / cesium chloride, etc., and use oligo d T cell mouth column method to extract poly A (+) RNA or mRNA If necessary, further synthesize cDNA and cRNA from mRNA (for example, F. M. A usubel et al., 'Short Protocolsin Molecular Biology (3rd edition) AC ompendiumof Methods from Curren Protocolsinolecular Biology) , 1 995, John Wiley & Sons, Inc. (USA)).

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

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

Specifically, when the cancer metastasis-related gene marker consists of the base sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient has a better postoperative prognosis or is less likely to metastasize. Identified as a patient. 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 Identified as a patient with a worse postoperative prognosis or with a higher likelihood 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 metastasis potential of cancer patients in vitro, which method comprises the steps of: SEQ ID NO :! Measure the expression level of at least one of the cancer metastasis-related gene markers including the nucleotide sequence shown in -45 or a mutant sequence thereof or the transcription or translation product level thereof using a probe corresponding to the marker, and This includes predicting the prognosis or metastasis after surgery using a significant level difference from a non-cancerous control sample as an index. The probe used in the present invention is not particularly limited as long as it can detect the above marker, but is usually a polynucleotide or an antibody. Therefore, a composition comprising such a polynucleotide or antibody can be used to predict the postoperative prognosis or metastatic potential of cancer patients.

2-1 Polynucleotide probe

 Specifically, the polynucleotide probe according to the present invention includes (i) a polynucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 to 45, (ii) a polynucleotide complementary to the polynucleotide of (i), (Iii) A polynucleotide variant of (i) and (ii), a polynucleotide of (iV) (i) or a polynucleotide of (ii) or a polynucleotide of (iii) hybridized under stringent conditions And (V) at least one, preferably at least 2, more preferably 3-4 selected from the group consisting of a fragment comprising 15 or more contiguous bases of said polynucleotide or variant. Includes 5 probes, for example 4-4 5 or 5-4 5 probes. The cancer metastasis-related gene markers according to the present invention can be divided into the following two groups 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 postoperatively of cancer patients The prognosis is better, and the marker involved in this group is the gene group of SEQ ID NOs: 28-45.

As used herein, “good prognosis” means that cancer is highly invasive, metastatic, or advanced, and the proportion of 5-year survivors of cancer patients determined in this way is about 7 0% On the other hand, “poor prognosis” 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. is there.

 In the present invention, at least one of the genetic markers belonging to each group, preferably at least 2, more preferably 3 to all, for example, 4 to all, 5 to all markers, Inspect with the corresponding probe.

 Probes for detecting markers belonging to Group 1 include polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 27, complementary polynucleotides, mutants thereof, and stringent genes. Probes selected from the group consisting of polynucleotides that hybridize under conditions, or fragments thereof containing 15 or more contiguous bases are included.

 On the other hand, a probe for detecting a marker belonging to Group 2 includes a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 28 to 45, a complementary polynucleotide thereto, a variant thereof, and a string thereto. Probes selected from the group consisting of polynucleotides that hybridize under harsh conditions, or fragments thereof containing 15 or more contiguous bases are included.

 Thus, the present invention also includes a composition containing each of the groups related to Group 1 and Group 2.

 In the present invention, a variant as a probe is one or more, preferably one or several, in the base sequence represented by SEQ ID NOs: 1 to 45, as defined above, such as substitution, deletion, addition, insertion, etc. Mutants having mutations, mutants comprising a sequence having identity of usually 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, 98% or more, etc. including.

Furthermore, in the present invention, a polynucleotide that hybridizes under stringent conditions hybridizes to the polynucleotide or variant of (i) to (iii) above under the hybridization conditions as defined above. If it is possible, there will be no particular limitation. Depending on the individual of the patient, the base sequence represented by SEQ ID NOs: 1-45 may have a gene that has been altered by biological events such as sudden mutation, polymorphism, or alternative splicing. The polynucleotide 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 number of bases. Fragments can have 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, 20 bases or more The number of bases, such as 2500 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 having about 100 bases or less, it can be synthesized using a DNA automatic synthesizer (for example, Applied biosyms, USA) using the phosphoramidite method.

 Alternatively, the polynucleotide can be produced by cDNA cloning. Obtain total RNA from the target cancer tissue, obtain poly A (+) RNA by oligo d T cellulose column treatment, and then c DN A live by reverse transcriptase-polymerase chain reaction (RT-PCR) method Create a rally from this library and register it in a data bank such as Gen Bank or ni Gene

By hybridization with a probe (15 or more, preferably 30 or more, more preferably 50 to 100 or more bases. Length) prepared in advance based on (Table 1 and Table 2) c You can get a DNA clue. The obtained clone is incorporated into an expression vector that is commercially available, and then introduced into an appropriate host cell such as Escherichia coli or Bacillus subtilis, and the host cell is propagated, or the sequence registered in the data bank. Polymerase chain reaction (PCR) using a primer prepared in advance based on the above (usually 15 to 30 bases, preferably 17 to 25 bases in length) and the cDNA clone as a saddle type Can be amplified. c For specific procedures and reagents for DNA cloning and PCR methods, commercially available kits, equipment, and reagents can be used. For example, Sambrook J et al., Mo.

1 e c u 1 a r C l o n i n g A L a b o r a t o r y Ma n u a l,

1 9 8 9 years, C o l d S p r i n g H a r b o r L a b o r a t o r y

P r e s s (USA); F. M. A u s b e l et al., S h o r t P r o t o c o

I sin Mo lecular B iology (3.) AC ompendiumof Me thodsfrom C urrent P rooco I sin Molecular Biology, 1 995, John Wiley & Sons, Inc. (USA).

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 fragment thereof against a polypeptide selected from the group consisting of the 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 against a polypeptide selected from the group consisting of the 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, or the like fragment.

 Therefore, 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 Is at least 2, more preferably 3 to all, eg 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 F ab, (F ab ′) ₂ , F c,

F c ', F d, F v, etc. are included. 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, horses, etc. Immunize animals such as sheep, goats, and hidges, and produce and purify antibodies against these antigens.

A polyclonal antibody immunizes the animal subcutaneously with an antigen of about 10 to 300 g, further boosts about 2 weeks later, collects blood about 3 weeks to 1 month after the first immunization, Ig G component containing the desired polyclonal antibody from serum, ammonium sulfate fractionation, ion exchange It can be made by a method that includes separation using chromatography. In order to increase specificity, the obtained IgG was 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 concentration buffer. A specific polyclonal antibody can be obtained by desalting by a method such as ultrafiltration. Antibody titers can be determined by conventional immunoassays, such as enzyme immunoassays (EIA, ELISA), radioimmunoassays (RIA), fluorescent antibody methods, luminescence immunoassays, etc. It can be measured by a conventional method such as a sandwich method, a latex aggregation method, a latex turbidimetric method, a red blood cell agglutination method, a latex agglutination method, etc.

 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, BalbZc mice) in the same manner as polyclonal antibodies, and boosted approximately 1 to 4 times at intervals of 1 to 4 weeks. Do. When the antibody titer reaches its peak, the antigen is injected intravenously or intraperitoneally for 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 (HG PRT) deficient cell line) to produce hybridoma cells and HAT (hypoxanthine, aminopterin). , Chimin) Make a selection. For cell fusion, antibody-producing cells and myeloma cell lines are mixed at a ratio of about 1: 1 to 20: 1 in animal cell culture media such as serum-free DMEM and RPM I-1640 media. In the presence of a cell fusion promoter such as polyethylene glycol. Confirmation of the target antibody can be performed by the immunoassay described above. Furthermore, in order to grow hyperidomas, about 10 million hybridomas are administered into the abdominal cavity of mice, and after the hybridomas are grown, ascites is collected 1 to 2 weeks later. Antibody purification can be performed by appropriately combining methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

An anti-peptide antibody is an antibody against a linear peptide on the surface of a protein, and can increase immunological specificity. Such peptides are for example K yte—Doolittle et al. Estimates of hydrophilic / hydrophobic regions, Em ini et al., probability of being located on the surface of a specific peptide site on a protein molecule, degree of bending of a polypeptide chain, eg C hou—Fasman It can be estimated by using a helix such as et al., Β sheet, secondary structure prediction of protein indicating turn, etc. alone or in combination. The estimated peptide can then be synthesized using a peptide synthesizer.

 Here, the synthesis of the target polypeptide (coded by the genes shown in Table 1 and Table 2 above) was carried out by incorporating the c DN clone into the expression vector and transformed or transfected with the vector. It can be obtained from the prokaryotic or eukaryotic host cell by culturing the cell or culture supernatant. Commercially available expression vectors can be used. Host cells include prokaryotic cells such as bacteria (eg, Enterococcus, Bacillus subtilis, Pseudomonas bacteria, etc.), yeast (eg, Saccharomyces, Pichia, etc.), insect cells (eg, S f cells), mammalian cells (eg, CHO). , COS, BHK, HEK 29 3 etc.). The vector consists of plasmids, cosmids, phages, etc., DNA encoding the target polypeptide, promoter, enhancer if necessary, polyaduration signal, ribosome binding site, replication origin, terminator, selection marker Can be included. In order to facilitate the purification of the polypeptide, a DNA sequence encoding a labeled peptide, such as a 6 to 10 residue histidine tag, a FLAG, a GFP polypeptide, etc., can also be included. The gene recombination techniques are described in Sambrook et al. (Above) and Ausbe1 et al. (Above), and the techniques described therein can be used for the present invention.

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

The sequence of the target polypeptide or a fragment thereof can be obtained by accessing the NCBI Home page based on the Gen Bank or Uni Gene registration 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 a microarray, that is, the probe is included in the form of a kit or a microarray.

 In the case of kits, polynucleotides or antibodies capable of detecting the total number of markers from one or more of each of the two groups of genetic markers (Tables 1 and 2), individually or in combination of two or more It can be packaged in a suitable container.

The antibody is a polyclonal antibody, a monoclonal antibody, an anti-peptide antibody or the like produced by the above method, but is not limited thereto. The type of antibody may be any type, class, or subclass, for example, IgG, IgM, IgE, IgD, IgA, IgGl, IgG2, IgG3, Including I g G 4, I g A l, I g A 2 and the like. Antibody fragments include F ab, (F a b ′) ₂ , F c, F d, F v and the like.

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

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

 Each of these microarrays is bound with the above-mentioned polynucleotide as a probe or the above-described antibody or fragment thereof. That is, on the surface of the array, one of the above gene markers or a transcription or translation product can be detected, a polynucleotide that can be hybridized with a gene marker or a variant thereof, or a polynucleotide encoded by these genes. An antibody or fragment thereof that can specifically bind to a peptide or a variant or derivative thereof is bound as a probe.

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

Variants are generally 80% or more, 85% or more, preferably 90% or more at the nucleotide or amino acid level with the complete or partial sequence of the above gene or polypeptide. More preferably, it has 95% or more identity and 98% or more identity.

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

3. How to predict prognosis or metastatic potential

 The method of the present invention is a method for predicting the postoperative prognosis or metastasis potential of a cancer patient after surgery, wherein the base sequence shown in SEQ ID NOs: 1 to 45 in a biological specimen derived from the patient Alternatively, at least one expression level of a cancer metastasis-related gene marker containing the mutant sequence or its transcriptional or translational product level is measured using a probe corresponding to the marker, so that the prognosis after surgery is improved. Postoperative prognosis or metastasis is possible using the relative difference in level between patients with good or lower metastatic potential and those with worse postoperative prognosis or higher metastatic potential. Including determining gender.

 Here, the expression level is the transcription or translation product level for the expression of the above-mentioned 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 a specimen from a patient group with a later prognosis or a higher possibility of metastasis. By identifying the above genes that show differences in expression levels, the postoperative prognosis or metastatic potential of cancer patients can be predicted in vitro.

 According to the present invention, the difference in gene expression level can be made by measuring the presence or amount of the gene or its corresponding polypeptide 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 tissue or cells that have been removed by surgery, tissue or cells obtained by biopsy, and the like.

 In the following, these two different methods will be described in detail.

3.1 Methods using polynucleotides

In order to detect 45 gene markers related to the present invention, the above-mentioned polynucleotide hybridized with each marker is 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 number of genes to be detected The more it is, the better the prediction accuracy.

 Hybridization should be performed by microarray method, blotting method such as Northern or Southern blot, Northern or Southern hybridization method, insitue hybridization method, quantitative RT_PCR method, etc. Can do. 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. '

 Any type of substrate can be used for the DNA microarray as long as it can immobilize nucleic acid probes. The solid phase includes, for example, glass, polymer, and the like, and a spacer including a reactive group for covalently binding a nucleic acid can be introduced with a crosslinker. 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, for example, a method of spotting DNA using a high-density dispenser called a spotter or layerer, a droplet is ejected from a nozzle It can be carried out using methods such as an inkjet method.

 DNA microarrays are prepared by labeling nucleic acids such as DNA or RNA in biological specimens and cDNA and cRNA derived from them with fluorescent substances such as Cy dye (Cr3 or Cy5). Hybridize with the above probe. The fluorescence intensity is read using a laser scanning reader and the data is analyzed by a computer.

In the ^plot method, the nucleic acid probe of the present invention is ^combined with a radioisotope (for example: ^{ί 2} Ρ and

³⁵ S) and fluorescent substances (rhodamine derivatives, Cy dyes, etc.) and then labeled with DNA or RNA in biological samples transferred to polymer membranes such as nylon, cDNA derived therefrom, c RN Hybridize with nucleic acids such as A. The signal is detected using a radiation detector or a fluorescence detector and Measure the degree.

 In the quantitative RT-PCR method, primers are annealed with cDNA and PCR is performed so that the target gene region can be amplified using cDNA prepared from RNA in biological samples as a template. The detected double-stranded DNA is detected. The ability to pre-label primers with radioisotopes or fluorescent materials, or by electrophoresis of PCR products on agarose gel and staining of double-stranded DNA with ethimubu-mide etc. Can be detected and quantified.

 PCR conditions include, for example, denaturation: 92-94 ° C. for 30 seconds to 5 minutes; annealing: 50-55 ° C. for 30-60 seconds; extension: 68-72 ° C. It includes 30 to 40 cycles of reaction, with 30 seconds to 10 minutes as one cycle. Reverse transcriptase is a commercially available enzyme such as Super Script® III (Invitrogen, USA) ゝ AM VR everse 丄, ranscriptase, Promega, USA), M-ML V (RN ase H_) (Takara Shuzo) , Kyoto, Japan) can be used.

 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, 1 M sodium chloride Z0.5% (WZV) sarkosyl

Hybridization in Z30% formamide, 60 ° C, 17 hours, then

6 XSSC / 0. 0 0 5% (W / V) Triton X— 1 0 2 solution, once at room temperature for 10 minutes, and 0.1 XSS CZ 0. 0 0 5% (W / V ) Triton X—

Contains one wash for 5 minutes while keeping at 0-4 ° C in 10 2 solution. Alternatively, another hybridization condition may be, for example, about 45 to 50 at 2 to 6 XSSC followed by 0.2 to 2 at about 50 to 65 ° C. XS

S C / 0. 1 to: Wash with 1% S D S; or 6 min. After hybridization in 6 X S S C, Den har d t solution, 0.2% S D S at 60 to 65 ° C.

Includes washing with 0.2 XSSC, 0.1% SDS from 0 to 65. For conditions and methods of hybridization, see, for example, A usubel et al., Cur. ent Protocolsin Molecular Biology, 1 9 8 9, John Wiley and Sons, USA).

 3.2 Antibody method

 An alternative method for measuring the expression level of the gene is an immunological method.

 The antibodies produced as described above can be used for the detection of the target polypeptide or fragment thereof in a biological specimen.

 By creating protein microarrays with many antibodies bound on a microarray substrate, or by spotting many antibodies on a filter such as a PVDF membrane, Polypeptide can be detected or quantified. Or, conventional immunological methods such as enzyme immunoassay (ELISA, EIA), fluorescent antibody method, radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetric method, latex agglutination, latex ratio A target polypeptide or a fragment thereof in a biological sample can be detected or quantified by a turbidity method, a hemagglutination reaction, a particle agglutination reaction or a Western plot method.

 When performing the reaction on a solid phase, as a solid phase carrier, a polymer film (filter) such as polystyrene, polycarbonate, or polyethylene, a plate, a tube, a strip, a particle such as a latex, a magnetic material, etc. Is included. The solid phase can be physically or chemically performed. For chemical coupling, the solid phase can be treated with a reagent such as maleating reagent or cyanogen bromide, and functional groups that react with amino groups of proteins can be introduced 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 rust peroxidase and alkaline phosphatase, fluorescent substances such as fluorescein, rhodamine and derivatives thereof, luminescent substances such as luciferase and luminol, ³² P, ^{1 25} I, ³⁵ Radioactive isotopes such as S are included. Labeling includes, for example, the dartal aldehyde method, the maleimide method, the pyridyl disulfide method, the chloramine T method, and the Bolton Hunter method.

4. Screening for cancer metastasis inhibitors The present invention further relates to 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 of cultured cancer cells and A screening method for a cancer metastasis inhibitor comprising screening a candidate drug for inhibition or suppression of Z or invasive ability is provided.

 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 a transcription product thereof, or Screening candidate drugs for inhibition or suppression of motility and / or invasive capacity.

 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 known metastatic human cancer cell line, MeWo; Tumor 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, etc., which can be used in the present invention .

 In the present invention, the degree of inhibition or suppression of expression of the human cancer metastasis-related gene or its transcription product can be determined by a comparative experiment with a control to which no candidate drug is added. Expression levels were obtained from cancer cell lines by well-known methods (for example, extraction with phenol, blackform Z, soamyl alcohol, guanidinium Z, cesium chloride, etc., ethanol precipitation, oligo d T cellulose column chromatography, etc.). A hybridization method using fluorescent or radiolabeled probes for RNA or mRNA or poly A (+) RNA, or for cDNA synthesized from RNA by the reverse transcriptase — PCR (RT-PCR) method (For example, Northern hybridization, Southern hybridization, DNA microarray, yarn and fabric microarray, etc.). Alternatively, the expression level is measured by measuring the intracellular level of the polypeptide encoded by the human cancer metastasis-related gene by an immunoassay using an antibody against the polypeptide or a fragment thereof, Western high-pridition, etc. Can be determined indirectly.

The probe is a nucleotide sequence of SEQ ID NOs: 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 less. Above, DNA having a sequence consisting of 1550 or more, 2200 or more, and 2500 or more nucleotides. The probe is preferably a labeled probe bound with a fluorescent or radioactive label. Fluorescent labels include, for example, fluoresamine, P-damine, their derivatives, Cy3, Cy5, etc. Radiolabels include, for example, radioactive phosphorus or iow atoms.

 An immunoassay is an analysis method that uses an antigen-antibody reaction, and is 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. Can do. These methods are well known in the art and their conventional techniques can be used in the present invention.

 Human culture (metastatic) In cancer cells, 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 compared to the control without the candidate drug. The candidate drug can be identified as a cancer metastasis inhibitor. Candidate drug screening can also be found by examining inhibition or suppression of motility and Z or invasive potential of human cultured cancer cells, preferably metastatic cancer cells.

 In vitro motility or invasion of cancer cells can be performed using, for example, a transwell chamber culture system. In the motility assay, for example, an insert (made by Becton Dickinson) with a polyethylene terephthalate film having a small pore of 8 microns in diameter is inserted into a 24-well cell culture plate, and serum-free cells inside the insert are inserted. Inoculated with metastatic cancer cell lines in the medium, cultured for 24 hours, and then counted by counting the number of cells that have passed through the small pores and moved to the lower membrane (Kozaki K et al., Cancer Research) 6 0: 2 5 3 5-4 0, 2 0 0 0). The invasion 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: 2). 5 3 5-4 0, 2 0 0 0).

In the above assembly, by adding a candidate drug to the serum-free cell medium, a candidate substance that inhibits or suppresses the motility and Z or invasive ability of metastatic cancer cells is identified as a cancer metastasis inhibitor. sell. Candidate agents include, but are not limited to, small molecules, peptides, polypeptides, proteins, nucleosides, oligonucleotides, polynucleotides, nucleic acids (DNA or RNA), and the like.

 The polypeptide or protein as a candidate drug includes, for example, an antibody or a fragment thereof against the polypeptide or protein encoded by the base sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof. In addition, the nucleic acid includes a ribozyme capable of cleaving mRNA corresponding to the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, s iRNA (sma lin terf er RN RN), and the like.

 In order to select the sequence of the siRNA, for example, known knowledge for the 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 ′ end nucleotide of the antisense strand is A, U, etc. (DM Dy Kxhoorn et al., Nature R ev. Mo 1. C ell Biol. (2003), 7 7: 7 1 74-7 1 8 1; A. Kh vorova et al., C ell (2003), 1 1 5: 2 09— 2 1 6).

Further, 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. Ribozyme-cleavable target sequences are generally known to be sequences containing NUX (N = A, G, C, U; X = A, C, U), for example, GUC triplets. Since the above-mentioned candidate triplet exists in the corresponding mRNA sequence encoded by the nucleotide sequence of SEQ ID NOs: 1 to 27 of the target tumor metastasis-related gene in the present invention, the candidate triplet is Ribozymes containing a sequence complementary to the portion of the mRNA sequence to be cleaved can be used for cleaving the target mRNA. Such ribozymes include hammerhead type ribozymes. Hammer one heads type Ribozaimu includes areas nucleotide sequence, which may only form a cavity for capturing a stable Mg ² + ions when RN A to sensor-site bound constituting the sensor portion Nukure And a nucleotide sequence comprising a region that is complementary to the sequence surrounding the cleavage site of the target RNA.

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

Example 1

 (Establishment of highly metastatic human lung cancer cell line (L NM 3 5))

 N C I-H 4 60 cells (Am e r i c a n T y p e C u l t u r e C o

(Obtained from 1 lection) (parent strain) 1 X 1 0 ⁷ cells Z 0 0 μ 1 (RPMI 1

6 40 cell medium) was subcutaneously transplanted into SCI D mice (purchased from Shizuoka Experimental Animal Research Institute (Shizuoka, Japan)). 45 days after transplantation, the lung metastasis of cancer cells was confirmed, and cancer cells from lung metastases were transplanted subcutaneously in another mouse, and the cancer cells from lung metastases were cultured in culture medium (10% Primary culture was carried out in RPM I 16 40) with urine serum. The cells were transplanted subcutaneously into another mouse, and similarly, cancer cells from lung metastases were cultured. In addition, the cells after 2 times i n v i v o s e l c t i o n were transplanted under the skin of mice, and cancer cells from axillary lymph nodes were cultured in culture medium (R PM with 10% sushi serum)

I 16 40)). Subsequently, cloning was performed by limiting dilution using a 96-well plate, and N C I — H 4 6 0 _ L NM

3 5 was obtained (Fig. 1).

 Separately, a low-metastasis strain (N 15) was obtained from the parent strain N C I — H 4 60 cells by the limiting dilution method. Furthermore, by treating L NM 3 5 with a demethylating agent (5 —aza—2 '—deoxycytidine) (1 // 1 ^ for 2 4 hours) in the same culture medium as above, low Transfer strains (L 2 D 2 and L 2 D 3 A) could be obtained.

 For the LNM35 and parent strains obtained by the above procedure, examine the motility of cells and metastasis by invasion, and the strength of metastasis to axillary lymph nodes and lungs by subcutaneous transplantation into mice. It was.

The motility assay is a transwelch filter with a filter of 8 m. Using a bar, the number of cells migrated to the back of the filter after counting for 24 hours was counted.

 The invasion ability is measured using a transwell chamber in which the gel is coated on a filter with 8 μm micropores and counts the number of cells that have migrated to the back of the filter after 24 hours of incubation. I went by the procedure.

 Lymph node and lung metastases were determined by weight and number of masses, respectively. As a result, as shown in Fig. 2, the LNM3 5 strain showed the highest levels of both motility and invasion, and also showed high levels of lymph node and lung metastasis. I confirmed that there was. On the other hand, the parent strains showed very low motility, invasive ability, lymph node metastasis, and lung metastasis, confirming that they were low metastasis cancer cell lines. Example 2

 (Detection of novel cancer metastasis-related genes)

 ene F i 1 ter Human Microarrays Release I and Release II (registered trademark) microarray (manufactured by Invitrogen) can be used to generate genes between highly metastatic LNM3 5 and parent N 15 The detection of specific genes showing current differences was examined.

Specifically, in this microarray, 5 g of total RNA was caged, and an oligo-d T primer and Super Scriptl I reverse transcriptase were used to produce 100 μCi of [ ³³ P] d CT. Reverse transcription in the presence of P, and c DN A microa'ray (Gen F i 1 ter Human M icroarrays manufactured by Invitrogen)

R e lea ase I and R ele ase I I) were hybridized. 2 M u r e a, 0.1% S D S, 50 mM sodium phosphate buffer (pH 7.

0), 1 50 mM NaC 1, 1 mM MgCl ₂ and 0.2% Alk Phos

After washing with D 1 RECT ^{R 1} blockingreagent (Amersham Biosciences), the microarray was brought into contact with the imaging plate (Fuji Photo Film, Tokyo, Japan) for 2 hours.

By scanning the imaging plate using 000 ^R '' (Fuji Photo Film Co., Ltd., Tokyo, Japan), expression of all 8, 6 4 and 4 genes corresponding to 1]., 1. 6 8 spots The profile was acquired twice. (Bioinformatics analysis)

 LNM3 5 and N 15 are each 2 times, and in the total 4 times of expression profiles, spots whose expression level (scanned spot value) is 0.1 or less are excluded from analysis due to detection limit. did.

 Using the 1 o w e s s method, which is a non-linear correction method, four expression profiles were subjected to no r m a l i z e, and then the average value was determined for each of L NM 3 5 and N 1 5.

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

 Furthermore, in the spot that was extracted, the expression level of the two expression profiles of LNM35 was both higher than 1.0 in the spot whose expression was enhanced by LNM35. In the spot whose expression was enhanced by N 15, when searching for spots that met the condition that the expression values of the two expression profiles of N 15 were both greater than 1.0, 45 Spots corresponding to these genes were extracted.

Example 3

 (Patient case)

 50 patients with lung cancer were obtained from a file of patients who had successfully undergone curative resection at Thoracic Surgery (Nagoya, Japan) at Aichi Cancer Center. All tumor specimens were embedded in OCT compounds and stored at 180 ° C after obtaining the required approval from the formal review department and written 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 Ί μm. Every 10 sections were stained with Giemsa, and under the guidance of a pathologist, total RNA was extracted from an area where the majority of the tumor tissue was located using an RN easy kit (Qiagen, USA). Approximately 5 μg of total RNA was reverse transcribed in the presence of 100 μCi of [ ³³ P] d CTP using oligo-dT primer and Superscript II reverse transcriptase. Invitrogen's c DN A microarray (Gene F i 1 ter Human Microarrays Release I and Release II) was re-noised and the microarray was then imaged (Fuji Film) For 2 hours, and scanning the imaging plate with BA S 5 000 (Fuji Film), 2 expression profiles of all 1, 1, 16 spots corresponding to 8,644 genes were obtained. Acquired times.

 (Bioinformatics analysis)

 After correcting the expression profile obtained using the r a n k- i n v a r i ant method, the expression value was determined from the average of the two times.

 (Relationship between expression pattern of 45 metastasis-related genes and recurrence and death)

 Based on the similarity of 45 genes extracted from the comparison of LNM3 5 and N 15, data on 50 lung cancer patients who underwent lung cancer surgery at Aichi Cancer Center (Nagoya, Japan) was clustered. Analyzed (Figure 3A).

 The dots shown in red or green in the figure indicate the expression status of each gene in individual cases. 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. The dendrograms in the figure show similarity. If the degree of similarity is high, the branches indicating the connection of the dendrogram are short, and if the degree of similarity is low, the branch indicating the connection of the dendrograms is long. The cases with the highest similarity are linked in order, and are finally compiled into a single tree diagram.

 Clustering analysis was performed on 50 lung cancer specimens, and they were classified into two large groups. The group on the left is a lot of genes with high expression (red dot ド)

It was a gene whose expression was enhanced by LNM35. On the other hand, in the group on the right side, many genes with high expression were genes whose expression was suppressed by the strength LNM35. Here, force group Meyer survival analysis of the prognosis of these two groups with the left group as Fata 1 group and the right group as F avorab 1 e group showed a significant difference (P = 0. 02 34) A prognostic difference was detected (Fig. 3 B). As a result, LNM

35 The group of genes whose expression is enhanced in 5 has a relatively high expression (Fata 1 group) has a poor prognosis, and the group of genes whose expression is suppressed by LNM35 has a relatively high expression (F The prognosis of the avorable group was good, using data from human lung cancer cases.

Using data from two lung cancer patients obtained at Harvard University, class The results of the data analysis are shown in FIG. 4A.

 We analyzed the data obtained at other facilities and examined whether the same results as in Fig. 3 could be obtained. Since the data of Har V Ar d is different from our data acquisition method, we could obtain only expression information for 38 genes out of 45 genes. Using the expression information of these 38 genes, the same analysis as in Fig. 3 was performed.

 6 Two lung cancer specimens were classified into two groups, left and right, after clustering analysis. One group on the right side was a gene with high expression (red dot). On the other hand, in the group on the left, many genes with high expression were genes whose expression was suppressed by LNM35. Here, the right group! ^ Ata 1 group, the left group is the F avorab 1 e group, and a force plan-meier 1 survival analysis was performed for the prognosis of these 2 groups. With a significant difference (P = 0. 038 5) A difference was detected (Figure 4B). As a result, the prognosis of the gene group whose expression is relatively high in LNM3 5 (Fata 1 group) is poor, and the expression of the gene group whose expression is suppressed in LNM3 5 is relative. High group

The fact that the prognosis of the group (F a vor a b ele) was good was confirmed even using the data of human lung cancer cases obtained at Harvard University (USA).

 Fig. 5A shows the results of a cluster analysis using data from 79 breast cancer patients acquired at the National Cancer Institute.

 We analyzed the data obtained at other institutions and examined whether the same results as in Fig. 3 could be obtained for solid cancers other than lung cancer. The data from the National Cancer Institute of the Netherlands (Netherlands) differed from the data acquisition method of the present authors, so only expression information on 37 genes out of 45 genes could be obtained. Using the expression information of these 37 genes, the same analysis as in Fig. 3 was performed.

 A clustering analysis of 79 breast cancer specimens revealed that they were classified into two large groups: left and right. One group on the left is the power of many highly expressed genes (red dots)

It was a gene whose expression was enhanced by LNM35. On the other hand, in the group on the right, many genes with high expression were genes whose expression was suppressed by LNM35. Here, a force planner-one survival analysis was performed on the prognosis of these two groups, with the left group as the Fata 1 group and the right group as the F avorable group. Thus (P = 0. 0032) a prognostic difference was detected (Fig. 5B). As a result, the prognosis of the group in which the expression of the gene group whose expression is enhanced in LNM 35 is relatively high (Fata 1 group) is poor, and the expression of the gene group in which the expression is suppressed in LNM35 is relatively The prognosis of the high group (Fa V orab 1 e group) was good, and it could be confirmed using data from human breast cancer cases obtained at the National Cancer Institute of the Netherlands.

 (Multivariate analysis using C o X proportional hazards model)

 A prognostic index based on 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, according to the proportional hazard model of COX, age (63 years old / under 63 years old), sex (male / female), smoking History (no smoking history / Yes), histological type (squamous / non-squamous carcinoma), stage (P STAGE), metastatic signature (F avorab 1 e / F ata 1) Examination was performed by variable analysis. As a result, it was shown with statistically significant difference that only the term Θ of the metastasis signature by stage and 45 gene is effective as a postoperative prognostic factor (stage: P = 0.0 1 4, 45 gene transfer signature: P = 0.003). In the figure, 9 5% CI indicates the 95% confidence interval, and P value indicates the risk factor.

 (Example of identifying cancer with good or bad prognosis after surgery)

 To classify cancers with good or bad postoperative prognosis, the signal noise function (signal — to-noisemetrics, Liolub et al., Science, Vol. 28 6, pp 5 3 1 to 5 3 7, 1 9 99) Using. When the cancer case group is defined as c 1 ass 1 for cancer with good postoperative prognosis and c 1 ass 2 for cancer with poor postoperative prognosis, the weight S which is the signal-noise statistic is given by Is calculated.

^{S =} 〃 cla _SS 】 — _C | _{a SS} 2 ^σ _Γ Ι α _{5 5} 1 + σ _{ε ΐ 3 5 5 2} )

Here, for each gene, ci _ass i represents the average value of all expression intensity data of c 1 ass 1, ch _{ss 2} represents the average value of all expression intensity data of c 1 ass 2, and _classl represents c 1 The standard deviation of all expression intensity data of ass 1 is shown, and _{class 2} shows the standard deviation of all expression intensity data of _{class 2} . Next, a weighted vote for gene x is calculated using the following W eighted-Voting formula.

V _X = S (G _x -b J

Where V _x is a weighted vote for gene x, S is a weight calculated by the above equation, G _x is the expression intensity (or expression level) of gene X, and b _x is

b _x = 1 + μ 2) / 2

(Where μ 1 and μ 2 are the averages of the mean values of c 1 ass 1. and class 2, respectively), indicating the center of two groups (ie, the center of gravity). When the sum of V _x in each group is greater than 0 by the method of adding weights according to the deviation from the center of gravity, cancer is classified as class 1, and when the sum of V _x is less than 0, cancer is classified as It can be classified as class 2.

For example, for samples i, 2, 3, 4 and 5 belonging to c 1 ass 1 and samples 6, 7, 8, 9 and 10 belonging to c 1 ass 2, genes (Gene) A, B, C (and above) , Measure the expression intensity (or expression level) of genes (G ene) X, Υ, Ζ (genes characterized by poor postoperative prognosis) The average value () and standard deviation (σ) of the expression intensity of each gene of the five samples of the group are calculated, and the weight (S) and the center (b _x ) are calculated from the above formula.

For sample A and sample B to be typed, measure the expression intensity (or expression level) of each gene, calculate the weight (S), G _x — b _x from the above equation, and obtain each V _{x. Find} the sum of V _x of 6 genes.

As a result, samp I e A is identified as classi because the sum of V _x is greater than 0. Sample B is separated from class 2 because the sum of V _x is less than 0. Industrial applicability

The present invention can predict the possibility of cancer metastasis and prognosis in cancer patients, and thus can greatly contribute to cancer treatment planning and prognosis management. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. A method for predicting the prognosis or metastatic potential of cancer patients after surgery in vitro, 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 At least one expression level or transcription or translation product level of a cancer metastasis-related gene marker containing a marker is measured using a probe corresponding to the marker, so that the prognosis after surgery is better or the possibility of metastasis is increased. To determine the postoperative prognosis or metastatic potential using the relative difference in level between the lower patient group and the patient group with worse postoperative prognosis or higher metastatic potential. Including the above method.

 2. When the cancer metastasis-related gene marker is composed of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient is a patient with a better prognosis after surgery or a lower possibility of transfer. The method of claim 1, identified as:

 3. 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 has a worse prognosis after surgery or a higher possibility of metastasis. The method of claim 1, identified as:

 4. A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45, a complementary polynucleotide thereto, a variant thereof, a polynucleotide that hybridizes under stringent conditions thereto, or 1 The method according to any one of claims 1 to 3, wherein the method is selected from the group consisting of those fragments comprising 5 or more consecutive bases.

 5. An antibody or fragment thereof against the polypeptide selected from the group consisting of the polypeptide consisting of the nucleotide sequence shown in SEQ ID NOS: 1 to 45 or a polypeptide encoded by a variant thereof, or a fragment thereof. The method according to any one of claims 1 to 3, wherein

 6. The method according to claim 5, wherein the antibody is a polyclonal antibody, a monoclonal antibody or an anti-peptide antibody.

 7. The method according to any one of claims 1 to 6, wherein the cancer is a solid cancer.

8. The method according to claim 7, wherein the solid cancer is lung cancer or breast cancer.

9. A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45, Or at least one selected from the group consisting of a variant thereof, a variant thereof, a polynucleotide that hybridizes under stringent conditions thereto, or a fragment thereof comprising 15 or more consecutive bases. A composition for predicting postoperative prognosis or metastatic potential of cancer patients in vitro, comprising two probes.

 1 0. A polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 28 to 45, a complementary polynucleotide, a variant thereof, a polynucleotide that hybridizes under stringent conditions, or 1 A composition for predicting in vitro postoperative prognosis or metastatic potential of a cancer patient, comprising at least one probe selected from the group consisting of those fragments containing 5 or more consecutive bases.

 1 1. 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 under stringent conditions thereto, or 15 A composition for predicting postoperative prognosis or metastasis potential in cancer patients in vitro, comprising at least one probe selected from the group consisting of the above-mentioned consecutive base-containing fragments.

 1 2. From a 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 to 45 or a polypeptide encoded by a variant thereof, or a fragment thereof, or a fragment thereof A composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro, comprising at least one selected probe.

 1 3. An antibody or fragment thereof against a polypeptide selected from the group consisting of a polypeptide 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 at least one probe selected from the group.

14. A group consisting of an antibody or a fragment thereof against a polypeptide selected from the group consisting of the 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 in vitro the postoperative prognosis or metastasis potential of cancer patients, comprising at least one probe selected from:

15. The composition according to any one of claims 9 to 14, wherein the probe is included in the form of a kit.

 16. The composition according to any one of claims 9 to 11, wherein the probe is included in the form of a microarray.

 1 7. Inhibition or suppression of a cancer metastasis-related gene comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 27 or its transcription product using cultured cancer cells, and the motility and Z of cultured cancer cells and Z Alternatively, a screening method for a cancer metastasis inhibitor comprising screening a candidate drug for inhibition or suppression of invasive ability.

 1 8. Prepare cultured cancer cells, culture the cells in the presence of a candidate drug, inhibit or suppress the expression of the cancer transfer-related gene or its transcription product, and the motility of Z or cultured cancer cells and The method according to claim 17, comprising screening a candidate drug for inhibition or suppression of Z or invasive ability.