JP2003274981A - Protein overexpressed in cancer cells and gene encoding the same - Google Patents

Abstract

(57) [Summary] (With correction) [Problem] To provide a protein and a gene thereof that can be effectively used for cancer diagnosis and prognosis (recurrence / metastasis) diagnosis and treatment. SOLUTION: A protein comprising an amino acid sequence of a specific sequence or an amino acid sequence in which one or several amino acids are added, deleted or substituted, and which is overexpressed in cancer cells, Encoding genes, cancer diagnostic markers, cancer diagnostic methods or screening kits using them, and targeted therapies.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein specifically overexpressed in cancer cells and a gene encoding the protein. Furthermore, the present invention relates to uses such as cancer treatment methods, detection methods, and prognosis methods.

[0002]

2. Description of the Related Art Cancer is a disease caused by mutation of a gene. Due to the mutation of the gene, cancerous cells acquire different properties from normal cells, repeat unlimited proliferation, and form metastatic tumors.

Conventionally, excision, radiation, anticancer agents, immunotherapy and the like have been performed as methods for diagnosing and treating cancers having such properties. Recently, genetic engineering methods such as gene diagnosis and treatment have been used. Various studies have been conducted to elucidate the mechanism of action of cancer at the gene / protein level and to diagnose and treat cancer with higher accuracy.

For example, cancerous cells often express a unique protein. Therefore, it is possible to test for the presence or absence of tumor tissue by a blood test, an immunological test, or the like, using a protein specifically expressed in cancerous cells and its gene as a marker.

Various markers such as CEA, SCC, AFP, and CA19-9 have been identified as markers used in such tests and are used for cancer screening.

[0006] By the way, among cancers, liver cancer is one of the cancers with a high mortality rate and is still on the increase. Liver cancer often occurs in the liver that has become chronic hepatitis and cirrhosis due to infection with hepatitis virus.
In order to reduce the occurrence of such liver cancer and suppress the progression thereof, it is important to elucidate the molecular mechanism of the occurrence and progression of liver cancer as well as antiviral treatment and antitumor treatment.

[0007]

However, although various gene mutations have been reported so far in liver cancer, until the details of the molecular mechanism relating to carcinogenesis and progression of liver cancer have been elucidated. However, the underlying cure has not yet been established.

The present invention has been made in view of the above circumstances, and proteins involved in carcinogenesis and progression of various cancers including liver cancer, genes encoding the same, and further It is intended to provide a use for

[0009]

That is, the object of the present invention is to provide the following means.

(1) Including the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids are added, deleted or substituted,
And a protein that is overexpressed in cancer cells, (2) β-
Overexpression is induced by a catenin mutation, (3) is a protein according to (1), which is a G protein-coupled receptor, and has SH2 and SH3 domains at the C terminus (1) ) Described in the above,
(4) The protein according to (1), wherein transcription is enhanced by activation of the Wnt signal transduction system,
(5) The nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 1 in which one or several nucleotides are added, deleted, or substituted, and comprises the amino acid sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2. (6) Amino acid of SEQ ID NO: 2 which includes a protein having an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence and which encodes a protein that is overexpressed in cancer cells A sequence or an amino acid sequence of SEQ ID NO: 2 having an amino acid sequence in which one or several amino acids are added, deleted or substituted, and comprising a protein that is overexpressed in cancer cells Cancer diagnostic marker,
(7) A method for detecting cancer using a tissue collected from within a subject, which comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2
Cancer is detected by a diagnostic marker that comprises a protein that has one or several amino acids added, deleted or substituted in the amino acid sequence of A method for detecting cancer, comprising: (8) a method for diagnosing cancer using cells / tissues collected from a subject, wherein the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: Screening for a protein containing an amino acid sequence in which one or several amino acids are added, deleted or substituted in the amino acid sequence of 2 and showing overexpression in cancer cells, and determining whether the protein is expressed or not. (9) Amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 expressed in a cancer cell tissue of a subject, which is characterized by diagnosing based on
Specifically binds to a protein containing an amino acid sequence in which one or several amino acids have been added, deleted or substituted, and which is overexpressed in cancer cells. A method for treating cancer, which comprises using a compound, (10) a method for prognosis after cancer treatment, comprising removing a tissue of a cancer-treated part from the inside of a subject, and amino acid of SEQ ID NO: 2 in the molecule. Based on the presence or absence of expression of the protein, which is a protein containing an amino acid sequence in which one or several amino acids of SEQ ID NO: 2 is added, deleted or substituted, and which is overexpressed in cancer cells, is screened. A method for prognosis of cancer, comprising: (11) a method for treating cancer, which comprises one of the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence and those sequences. Alternatively, a method for treating cancer characterized by administering a suppressive compound that specifically binds to a plurality of genes including a gene consisting of all or a gene product thereof or directly or indirectly to the gene or the gene product. (12) Consists of at least two of the nucleotide sequences of SEQ ID NO: 3 to SEQ ID NO: 8, and one or several nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 1 are added, deleted or replaced. (13) a polynucleotide comprising a nucleotide sequence having a nucleotide sequence and its complementary sequence and used for the synthesis, amplification and detection of a gene and protein showing overexpression in cancer cells, It has all or part of nucleotides, and one or several amino acids of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 has been added or deleted. Is a protein quantification kit comprising a substituted amino acid sequence and quantifying a protein which is overexpressed in cancer cells, (14) the nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof and A cancer cell screening kit comprising a plurality of gene fragments containing a gene consisting of part or all of the sequence.

The present inventors collected and analyzed tissues of various cancer patients in order to study the development and progression of (liver) cancer, and found that a gene overexpressed in cancer cells at high frequency was found. I found it. In such cancer cells, mutation of β-catenin occurred in almost all cases. The cDNA was then isolated, cloned and its sequence analyzed.

A homology search of the obtained nucleotide sequence using BLAST revealed that there was a site having 100% homology with the gene sequence of accession number NM_003667. Also, Unige
It was also found to be Gpr49 registered in ne Database.

However, there has never been a view that the relationship between the protein and cancer was found, and the present inventors completed the present invention for the first time when the present inventors found a correlation with the expression in cancer. It is a thing.

In recent years, many studies have revealed that β-catenin is deeply involved in cell adhesion by cadherin and abnormality in Wnt signaling system. Cell adhesion is a function related to cancer cell metastasis, and Wnt signaling system is a function related to cell proliferation. It is considered that one of the causes of the canceration of cells due to a decrease or abnormality in these functions due to the mutation of β-catenin.

From the above, the protein of the present invention is
For example, by using it as a cancer-specific marker for cancer diagnosis or treatment, it becomes possible to perform more accurate diagnosis, treatment, or prediction than at present.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.

First, the protein of the present invention will be described.

The protein of the present invention contains the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been added, deleted or substituted, and can be used in cancer cells. On the other hand, it is a protein that is overexpressed.

Further, the protein of the present invention is characterized in that overexpression is induced by mutation of β-catenin in cancer cells.

The protein of the present invention is a G protein-coupled receptor and is characterized by having SH2 and SH3 domains at the C terminus.

The protein of the present invention is G protein-coupled 7
Transmembrane receptor, thyroid stimulating hormone receptor (TSHR), follicle stimulating hormone receptor (FS
HR) and luteinizing hormone receptor (LHR), etc., and belong to the glycoprotein hormone receptor subfamily.

Both SH2 and SH3 domains are functional domains involved in signal transduction. SH2 recognizes phosphorylated tyrosine and SH3 recognizes a proline-rich sequence. That is, it is considered that the protein of the present invention acts on a plurality of signal transductions related to various functions such as proliferation and differentiation.

Further, the present invention is characterized in that the Wnt signal transduction system is activated by the mutation of β-catenin, whereby transcription is enhanced. The Wnt signal transduction system is a transduction system involved in cell proliferation, and it is known that the cells become cancerous due to an abnormality in the system. Among these transduction systems, β-catenin is important. It has been shown to play a role. Mutation of β-catenin leads to the same situation as activation of the Wnt signal transduction system, and it is considered that transcription of downstream genes including the protein of the present invention is enhanced.

From this, the protein of the present invention is
It can be said that it is useful as a marker for detecting an alteration in the t signal transduction system and a β-catenin mutation.

The protein of the present invention can be used by isolating and cloning it from human organs, cell lines and the like by a conventionally known method.

When used as a peptide, it can be chemically synthesized according to the amino acid sequence of SEQ ID NO: 2 of the present invention.

The protein of the present invention is transformed by incorporating the gene according to the present invention described below into an appropriate vector using a conventionally known recombinant DNA technique and introducing the vector into an appropriate transformant. It is also possible for the body to produce proteins.

Further, since the protein of the present invention has a characteristic of being strongly expressed in cancer, it can be effectively used for diagnosis and treatment of cancer as described later.

The gene of the present invention is a gene encoding the protein of the present invention, which is obtained by adding, deleting or substituting the base sequence of SEQ ID NO: 1 or one or several bases of the base sequence of SEQ ID NO: 1. The nucleotide sequence of the protein of the present invention cD
NA total length is shown. Similarly, in FIGS. 1 to 7 showing the base sequence of the protein of the present invention (base sequence of SEQ ID NO: 1),
Accession number AF062000 in lower case
The same sequence portion as 6, and the capital letters represent the sequence portion identified by the present inventors.

The gene of the present invention is, for example, c according to the present invention.
The gene according to the present invention, which can be isolated from an existing genomic library using DNA or a partial sequence thereof as a probe, is a human prepared from a human cell using, for example, an oligonucleotide probe synthesized based on the nucleotide sequence. It can be obtained by screening a cDNA library. Alternatively, it is possible to synthesize this oligonucleotide using the PCR method as a primer.

In general, a gene is found to have polymorphism due to individual differences, and therefore, a gene having one or more bases added, deleted or substituted is naturally included in the present invention. And
A protein consisting of amino acids in which one or more amino acids are added, deleted or substituted resulting from these changes is also included in the present invention as long as it has the activity of the protein having the amino acid sequence of SEQ ID NO: 2. .

Further, all or a part of the gene of the present invention or a sequence complementary thereto can be used as a probe for cancer gene diagnosis. Furthermore, a kit containing a plurality of these fragments can be prepared and used for detection and diagnosis of cancer.

For example, the nucleotide sequences of SEQ ID NOs: 3 to 8 can be used as a primer for the protein of the present invention. Of these, SEQ ID NOS: 3 and 4 are used as forward primers, and SEQ ID NOS: 5 and 6 are used as reverse primers in the PCR method to quantify the protein of the present invention. Further, SEQ ID NOS: 7 and 8 can be used as primers for the mouse homologue of the present invention to quantify the protein of the present invention in the homologue.

Next, applications of the protein and gene of the present invention will be described.

The cancer diagnostic marker of the present invention comprises the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids are added, deleted or substituted, and It has a protein that is overexpressed in cells. As described above, not only the gene diagnosis is performed using this marker as a probe, but the protein to be diagnosed is isolated from the tissue to be diagnosed by a conventionally known method, and the presence or absence of the expression of the protein of the present invention is screened. It is also possible to measure and measure the expression level to detect and diagnose cancer.

For example, since the protein of the present invention is overexpressed on the cell membrane peculiar to cancer cells, it is possible to target the protein and treat with a compound that specifically binds to the protein. It becomes possible to efficiently treat cancer cells.

Examples of the compound that specifically binds to the protein of the present invention include antibodies, antagonists and agonists of the protein.

The antibody according to the present invention can be obtained as a monoclonal antibody using the protein of the present invention as an antigen, using a hybridoma produced by a conventionally known method.

The antagonist and agonist according to the present invention may be DNA, protein or non-protein.

In addition to the missile therapy, the protein of the present invention is used as a direct target to modify it by substituting, adding or deleting a part of the gene of the present invention to inhibit the function of the protein. It is also possible to treat cancer.

Further, by screening the protein of the present invention and diagnosing based on the presence or absence of the expression of the protein, not only detection and diagnosis of cancer but also recurrence diagnosis after removal of cancer tissue by surgery ( Prognosis).

Further, in the diagnosis of cancer cells, it is also possible to perform the diagnosis by screening the genes in the test tissue using a screening kit equipped with a plurality of gene fragments containing the gene of the present invention.

As described above, according to the protein, gene of the present invention and the use for cancer treatment using these, it becomes possible to perform cancer treatment with higher accuracy.

[0044]

The present invention will be described in detail below with reference to examples. However, it goes without saying that the present invention is not limited to this.

<Material Adjustment> 38 hepatocytes from patients (average age 62.6 years, 20-76 years old) who underwent resection surgery at the National Cancer Center Hospital from July 1998 to June 1999. Cancers (HCC) and their corresponding non-cancerous liver tissues were collected and used for analysis.

The normal liver was collected from a colorectal cancer patient whose liver had metastases. The tissue was immediately cryopreserved and stored until use.

HCC, non-cancerous tissue and normal liver tissue were judged histologically. Clinicopathological features are shown in the table of FIG. For histopathological grade and HCC macroscopic type classification, liver cancer handling agreement (3rd edition)
Classified according to. Vascular invasion was defined including both tumor invasion into the portal vein and intrahepatic metastasis. In the case of multicentric HCC, the largest nodule was used in the following analysis. Human HCC cell lines PLC / PRF / 5 and HepG2 are A
Obtained from TCC. KYN-1, KYN-2 and KI
M-1 used was that provided by Masamichi Kamishiro of Kurume University. Other HCC cell lines Li7, Li21, Li
24, tPH5T and the corresponding non-neoplastic hepatocyte cell line tPH5CH, and mouse hepatocyte MHT were established by the present inventors. tPH5T, tPH5CH and MHT
Was immortalized with SV40 large T antigen.

All cell lines were 10% as described above.
Fetal bovine serum, 100 U / ml penicillin and 100 μ
RPMI-16 supplemented with g / ml streptomycin
The cells were cultured in 40 medium.

<RNA preparation> Acid Guanidin
ium thiocyanate-phenol-ch
Total R using the loroform method (AGPC method)
The NA was adjusted.

0.1 of each of the above 38 tissue sections
~ 0.2g to 4M guanidine thiocyanate, 25m
0.5 ml of a mixed solution of M sodium citrate (pH 7.0), 0.5% sodium N-lauroyl sarcosine and 0.1 M 2-mercaptoethanol was added and homogenized with a homogenizer, and the homogenate was added to a 1.5 ml Eppendorf tube. Moved to.

To this, 1) 2M sodium acetate (pH
4.0) 50 μl, 2) Phenol 0.5 ml, 3) Chloroform / isoamyl alcohol (49/1) 100
μl were added sequentially, mixing 2-3 times each time, and finally 1
The mixture was vigorously mixed for 0 seconds and then ice-cooled for 15 minutes.

Then, after centrifugation at 10,000 × g for 20 minutes, the aqueous layer was transferred to another tube. 0.5 ml of isopropanol was added to the aqueous layer, and the mixture was left at -20 ° C for 1 hour or longer.

RNA was then precipitated by centrifugation. Furthermore, the precipitate was washed with 80% ethanol, dried, and then dissolved in autoclaved distilled water to obtain total RNA.

<MRNA differential
Display PCR (mRNA DD-PCR)> The thus obtained total RNA was subjected to the mRNA DD-PCR method, which is one of the methods for comprehensively analyzing the difference in gene expression among a plurality of samples.

2.5 μg RNA in a 0.5 ml tube,
Anchor primer (50 pmol) was added, the total amount was made 10 μl with DEPC-treated water, and the mixture was heated for about 10 minutes by a thermal cycler heated to 70 ° C. After heating, the tube was rapidly cooled and centrifuged lightly. 2 x RT mix in tube (2 x Nμl DEPC treated water, 10 x PCR buffer 2 x Nμl, 25mM MgCl2 2 x Nμl, 0.
1M DTT 2 × Nμl, 10 mM dNTP 1 ×
Nμl, Super Script II 1 × Nμ
l) was added and mixed. Set the tube in the thermal cycler cooled to 25 ℃ and
Minutes, 42 ° C. for 50 minutes, 70 ° C. for 15 minutes. After that, the tube was lightly centrifuged, and 80 μl of TE was added and mixed well to obtain a cDNA solution.

CDNA solution thus obtained 2.
0 × Nμl, dH ₂ O 13.0 × Nμl, 10 × Gen
eTaq buffer 2.0 × Nμl, 2.5 mM dNT
P1.6 × Nμl, anchor primer 0.2 × Nμl
To prepare a PCR mix.

1 μl of an arbitrary primer was dispensed into each well or tube. 0.1 for each PCR mix
× Nμl GeneTaq and normal Taq polymerase were added and mixed. Then 1 for each well (tube)
The PCR mix was added in 9.0 μl increments. The plate was set on a thermal cycler and reacted as follows. (94 ° C x 3 minutes + 40 ° C x 5 minutes + 72 ° C x 5 minutes) x 1 cycle (95 ° C x 15 seconds + 40 ° C x 2 minutes + 72 ° C x 1 minute) x 2
0 to 25 cycles 72 ° C. × 5 minutes 5 μl of the reaction product obtained as described above was subjected to polyacrylamide electrophoresis, and a band showing an interesting difference in expression level was cut out from the obtained band.

Half of the gel piece thus obtained was transferred to a 0.5 ml tube, and in a system of 50 to 100 μl,
PCR was performed under the following conditions to re-amplify the band. 94 ° C x 3 minutes x 1 cycle (95 ° C x 15 seconds + 40 ° C x 2 minutes + 72 ° C x 1 minute) x 1
~ 20 cycles 72 ° C x 5 minutes About 5 µl of the above PCR reaction product was added to HA-Yellow (T
Agarose) -containing agarose gel was electrophoresed, the band of interest was cut out, re-amplified under the same PCR conditions, and then electrophoresed on HA-Red (Takara) -containing agarose gel for cloning.

<Northern Blot Analysis> Using 15 μg of total RNA of each example prepared as described above, 1.0% agarose formaldehyde denaturing gel electrophoresis was performed. Then, using the capillary blotting method, Hybond
It was transferred and fixed on a -N ⁺ membrane (Amersham). After fixing to the membrane, prehybridization was performed to hybridize the Gpr49 DNA probe with glyceraldehyde phosphate dehydrogenase (GAPDH). After hybridization, the membrane is washed with 0.1 × SSC
/ Washed with 0.1% SDS solution twice at 65 ° C for 30 minutes. Then, autoradiography was performed at −80 ° C., and the reaction was quantified with a densitometer.

Clone 141 was obtained from the clones obtained as described above. The result is shown in FIG. Figure 9
Is a Northern blot of clone 141 and GAPDH cDNA (control). In addition, FIG. 10 shows the expression level of mRNA of clone 141 in real time RT-P.
It is a graph quantified by CR and standardized by GAPDH.
The vertical axis represents the expression ratio of the protein of the present invention to GAPDH, and the horizontal axis represents each cell line 1: PLC / PRF / 5, 2:
KYN-2, 3: Li7, 4: KIM-1, 5: Hep
G2, 6: Li21, 7: Li24, 8: tPH5T
H, 9: tPH5CH, 10: KYN-1.

<Cloning of Gpr49> BLAST was obtained only by analyzing the clone 141 obtained as described above.
Since sufficient information cannot be obtained from the above, further screening / cloning was performed using a cDNA library.

Preparation of cDNA library The mRNA derived from PLC / PRF / 5 cell line is ethanol precipitated. After washing the precipitate with 70% ethanol, 1
Dissolve in 5 μl of 5 mM Tris-HCl (pH 7.5). Then, after keeping the temperature at 65 ° C for 5 minutes, the temperature is transferred to 37 ° C. Then, a solution having the following composition is added. 10 × reverse transcriptase buffer 3.0 μl 2 mM dATP, dGTP, dTTP 3.0 μl 20 mM [α- ³² P] dCTP 3.0 μl 1.2 μg / μl vector primer 3.0 μl 15 units / μl RTase 3.0 μl H ₂ O 2.3 μl After adding this solution, incubate at 37 ° C. for 30 minutes. After confirming TCA precipitation, a liquid having the following composition is added to the remaining solution and stirred. 0.25 M EDTA 3.0 μl 10% SDS 1.5 μl Next, 35 μl phenol / chloroform was added, and the mixture was well stirred and then centrifuged for 1 minute. To the supernatant, 35 μl phenol / chloroform was added, stirred well, and then centrifuged for 1 minute. 35 μl of 4 M ammonium acetate and 1 in the supernatant
Add 40 μl ethanol and leave for 15 minutes on a dry ice / ethanol mixture or dry ice. Then, the tube is left to stand at room temperature for 1 to 2 minutes, and when the turbidity of the precipitated nucleotide disappears, lightly stir it for 15 minutes.
Centrifuge for minutes. Then, after removing the ethanol, 35 μl of TE was added to the precipitate and stirred, and then 35 μl of 4M
Ammonium acetate and 140 μl of ethanol are added to perform ethanol precipitation. After removing ethanol, perform ethanol precipitation again. The precipitate thus obtained is 70
Wash with% ethanol.

A solution having the following composition was added to the precipitate, and the mixture was stirred and then kept at 37 ° C. for 5 minutes. H ₂ O 12 μl 10 × TdT buffer 2 μl 0.15 μg / μl Poly A 2 μl 1 mM DTT 2 μl 2 mM [α- ³² P] dCTP 0.6 μl Millipore filter (0.
0.5 μl of the sample prepared as described above is sampled and spotted on a 45 μm Type HA) and quickly sucked. This is washed twice with 3 ml of 1N HCl, 0.1 mM sodium pyrophosphate solution. 30 units Td
After adding T, the mixture was kept at 37 ° C. for 5 minutes and then left on ice.
After checking the TCA precipitation, the following solution is added and stirred. 0.25 M EDTA 2 μl 10% SDS 1 μl Next, 20 μl of phenol / chloroform is added, and the mixture is stirred and then centrifuged. To this supernatant, 20 μl of 4M ammonium acetate and 80 μl of ethanol are added, the same treatment as the above ethanol precipitation is performed, and the precipitate is washed with 70% ethanol.

25 μm was added to the precipitate obtained as described above.
l water and 3 μl HindIII buffer are added and stirred. Then add 14 units of HindIII, 37
Incubate at ℃ for 1 hour. After that, 1 μl of the sample was taken out and subjected to 1% agarose gel electrophoresis to perform HindIII.
Check disconnection.

To the remaining sample, 3.0 μl of 0.25
After adding M EDTA, 1.5 μl of 10% SDS,
Add 35 μl of phenol / chloroform, stir and centrifuge. Phenol / chloroform is added again to the supernatant produced by centrifugation, and the mixture is stirred and centrifuged to collect the supernatant. To this supernatant, 35 μl of 4M ammonium acetate and 140 μl of ethanol are added to perform ethanol precipitation. 35μ in the supernatant
TE of 1 is added and dissolved, and ethanol precipitation is performed again.
The generated precipitate is washed with 70% ethanol, the precipitate is well dissolved in 10 μl of TE, 10 μl of ethanol is added, and the mixture is stored at −20 ° C.

Sample 1.0 obtained as described above
0.5 μl (0.08 pmol) of 0.15 pmol / μld G-linker, 2.0 μl of 5 × hybridization buffer, and 6.5 μl of H ₂ O were added to μl,
Stir. Then, the temperature is kept at 65 ° C. for 5 minutes. Then, the tube containing the sample is transferred to an incubator at 43 ° C. and kept warm for 30 minutes. Further 18.0 μl of 5 × ligase buffer, H ₂ O 70.7 μl, 10 mM βNAD
Add 1.0 μl and cool on ice for 10 minutes. 0.6μ
g of E. coli DNA ligase (TAKARA) is added, and the mixture is gently stirred and then reacted overnight at 12 ° C. Next, the following solution and enzyme are added. 2 mM dATP, dGTP, dCTP, dTTP
2.0 μl 10 mM βNAD 0.5 μl E. coli DNA ligase 0.6 μl (TAKAR
A60 unit / μl) E. coli DNA polymerase 0.5 μl (TAK
ARA6 unit / μl) RNaseH 0.2 μl (TAKARA60 unit / μl) Then, gently stir and react at 12 ° C. for 1 hour, and transfer the tube to room temperature (about 22 ° C.) for 1 hour. Then 0.5 μl is withdrawn, diluted with 50 μl TE, transfected into E. coli (DH5) competent cells and the size of the cDNA library is measured. Dispense 200 μl of each competent cell into a 15 ml plastic tube placed in ice water. Dispense 3 to 5 μl of each of the above-mentioned samples into each competent cell, and leave on ice for 30 minutes. After incubating at 42 ℃ for 30 seconds,
Place in ice water for 1-2 minutes. 0.8 ml in each tube
SOC is added and shaken for 1 hour at 37 ° C. Then 1 liter
LB (containing 50 μg / ml of ampicillin) containing all the above samples. After stirring, 100, 30, 10, 2
Remove 3 µl of top agar that had been kept warm at 47 ° C, and sprinkle on L plate (containing ampicillin). The rest is shake-cultured at 37 ° C. overnight. L plate is also 3
Incubate overnight at 7 ° C. Take 93 ml of shake-cultured LB and mix with 7 ml of DMSO.

Screening Using the cDNA library obtained as described above, screening was carried out by a conventional method. A replica filter was prepared by a conventional method, and after performing prehybridization and hybridization, clones showing a positive reaction were selected and isolated.

Plating 0.5 ml plating bacteria and 2-3 × 10
After adding ⁴ pfu of phage and mixing, and further adding 7 ml of 0.7% top agarose, it is spread on a 150 mm LB plate and spread on the whole. Once the agarose has set, invert the plate and incubate in a 37 ° C. incubator until the plaques are fluent. The plate is then placed at 4 ° C for 1 hour or more to cool.

Then, a nitrocellulose membrane is placed on the plate and the plaque is transferred.

The transferred film was washed with 0.2 M NaOH / 1.5.
The sample was placed on a filter paper soaked with M NaCl for alkali treatment, and then 0.4 M Tris-HCl (pH 7.
6) Put it on the filter paper soaked with / 2xSSC and return to neutral. After drying the membrane, at 80 ° C for 1.5-2 hours or 4
Heat at 2 ° C. overnight to fix DNA to filter.

Using the replica filter thus obtained, prehybridization and hybridization are carried out by a conventionally known method. The plaques showing a positive reaction in this manner are collected, placed in a suspension medium containing chloroform, and plated on 3 to 6 plates so that the density becomes about 500 plaques or less per 100 mm plate.

Replica filter preparation, prehybridization, and
Hybridization is performed, and plaques showing a positive reaction are selected again. The same procedure was repeated again to select only plaques showing a positive reaction, and the above procedure was repeated until only a single clone was obtained, and a desired clone was selected.

Thus, a 2324-bp clone was obtained, and its sequence was analyzed by BLAST. The 5'-terminal 846-bp sequence of the clone has 100% homology with the BLAST accession number NM_003667 orphan G protein-coupled receptor, and is a gene registered as Gpr49 in the Unigene database. It turned out that

The nucleotide sequences of the protein of the present invention (Gpr49) obtained as described above are shown in FIGS. The lower case part shows a part having 100% homology with NM_003667, and the upper case part shows the part identified by the inventors in the present experiment. <Real-time quantitative RT-PCR> Next, SYBR green PCR core reagent kit (Perkin-Elmer A
R of the protein of the present invention by RT-PCR according to a conventional method using the Applied Biosystems).
Expression analysis of NA was performed.

CDNA of each tissue sample and cell line was prepared by using oligo dt primer and AMV reverse transcriptase XL (TaKa
Ra) was used to prepare from the conventional DNase-treated total RNA. Primer expression software (Perk
in-Elmer) was used to prepare the following primers for Gpr49. GSP1 (forward): 5'-CTCGTGGGCCC
CCTACTTC-3 'GSP2 (forward): 5'-gaggatctgg
tgagcctgagaa-3 'GSP3 (reverse): 5'-cataagtgatg
ctggagctgggtaa-3 'GSP4 (reverse): 5'-TCATGGTCCCTT
AATGTTACTGAACC-3 ′ Further, the following primers were prepared as primers for FEX which is a mouse homolog of GPR49. GSP5 (forward): 5'-GAGTCAACCCA
AGCCTTAGTATCC-3 'GSP6 (Reverse) 5'-CATGGGACAAAT
GCAACTGAAG-3 'Among the primers obtained as described above, GSP
2, 3, 5 and 6 were used for real-time quantitative RT-PCR. In addition, the expression of GAPDH was quantified as a control in order to standardize the amount of RNA.

In addition, the unpaired t test was used to analyze the difference in the expression level of the protein of the present invention in HCC and control non-cancer liver tissues. In addition, the association between Gpr49 expression and clinicopathological parameters was analyzed using χ2 test and Fisher's exact test, and StatView.
(Ver.5.0) (Abacus Concept
s, Berkeley, CA, USA). A P value of 0.05 or less was determined to be significant.

The results are shown in FIG. FIG. 11 is a graph showing the RNA expression level of the protein of the present invention in each sample. The left side of the graph shows the expression amount of RNA of the protein of the present invention in hepatocytes, and the right side thereof shows the expression amount in non-cancer liver tissue or normal liver tissue.
As can be seen from this graph, RNA of the protein of the present invention was expressed in many cancer tissues, but hardly expressed in normal tissues.

Further, in order to examine the overexpression of RNA of the protein of the present invention in HCC, the expression level ratio (T / N ratio) in tumor and non-cancer liver was calculated. Overexpression was judged when the T / N ratio exceeded 3.
The results are shown in the table of FIG. 12 and the table of FIG. As shown in the figure, 18 cases (47%) out of 38 cases with T / N> 3 in cancerous tissues, all of which were classified as overexpression.

<Correlation with β-catenin> PCR and β-catenin sequence analysis In PRF / PLC / 5 and HepG2 in which RNA of the protein of the present invention is highly overexpressed, β-catenin is cytoplasmic and nuclear. It has been shown that
-It was thought that it might be related to the mutation of catenin. Therefore, in order to investigate the correlation between the protein of the present invention and the mutation of β-catenin, the following experiment was conducted.

Glycogen synthase kinase-3β
It is thought to be involved in phosphorylation of (GSK-3β) 4
Exon 3 of β-catenin containing one site was amplified by PCR using the cDNA of each sample obtained in the above Preparation Example.

For PCR, the exon 2 forward primer of β-catenin (5'-CCAGCGTGGACA
ATGGCTA-3 ′) and β-catenin exon 4 reverse primer (5′-TGAGCTCGAGTCA
TTGCATAC-3 ') was used.

After confirming the PCR product by 2% agarose gel electrophoresis, sequence analysis was carried out from both directions using the primers used in PCR.・ Preparation of β-catenin mutant strain and P-catenated wild-type β-catenin cDNA containing all retroviral infection coding regions
Amplified by CR, pBluescript (Strata
gene) and subcloned. A β-catenin mutant strain in which Thr-41 and Ser-45 corresponding to the GSK-3β phosphorylation site are changed to alanine is QuickC.
change Site-Directed Mutage
It was created using the nessis Kit (Stratagene). Subsequently, the wild-type β-catenin and β-catenin mutant strain were added to the retrovirus expression vector pLNCX2.
The recombinant retrovirus subcloned into (Clontech) was used as a FuGENE6 transfection reagent (Boe) into 293 cells of pLNCX2 and pCL-Eco into which the wild type and mutant β-catenin were incorporated.
ringer Mannheim, Indiana
polis, IN). 24 hours after transfection 293
The supernatant of individual cells was collected, filtered through a sterile 0.45-microfilter, transferred as a retrovirus-containing supernatant onto MHT cells supplemented with 8 μg / ml polybrene, and infected.

24 hours after infection, the infection medium was treated with 10% FC
The medium was replaced with 1640 medium supplemented with S (fetal bovine serum). 48 hours after infection, the cells were treated with geneticin 500 μg /
Divided into medium containing ml. Then, total RNA was extracted from the resistant clone and real-time quantitative RT-PC was performed.
It was analyzed by R.

The above results are shown in FIGS. 11 and 13 to 15. In the example of FIG. 11, the example in which exon 3 of β-catenin undergoes somatic mutation is marked with * (16 out of 38 cases: 42%). Two variants were found in Examples 33 and 40. In Example 8, when exon 3 was amplified by PCR, the band was separated into two bands (data omitted). Analysis of this band revealed a deletion of 51-bp. 13 and 14 are electropherograms showing missense mutations and amino acid substitutions. In most cases, residues 33, 37, 41 encoding serine or threonine
Or an amino acid substitution occurred at 45 or occurred in these adjacent residues. The β-catenin mutation was frequently found in hepatocellular carcinoma in which the protein of the present invention is overexpressed (14 out of 18 cases: 78%). On the other hand, the β-catenin mutation in the cases where the protein of the present invention was not overexpressed was only 2 out of 20 cases (10%). FIG. 15 is a graph showing the effect of β-catenin on the expression of the protein of the present invention in mouse hepatocytes. It was found that the expression level of the protein of the present invention in the mouse β-catenin into which the mutant β-catenin was introduced reached about four times that in other hepatocytes into which other wild-type β-catenin or MOCKpLNCX2 was introduced.

From the above, it was found that there is a strong correlation between the overexpression of the protein of the present invention and the β-catenin mutation.

Further, the overexpression rates were found to be about 83% and 34% for females and males respectively (data not shown).
From this, it is considered that the protein of the present invention is involved in the difference in expression of cancer caused by the difference in sex.

[0087]

Industrial Applicability As described above, the protein of the present invention and its gene are strongly expressed in liver cancer cells, and β-
It has the property that overexpression is induced by mutation of catenin. By utilizing this characteristic, it can be effectively used for various purposes such as cancer diagnostic marker, target therapy, gene diagnosis and gene therapy, and other cancer recurrence diagnosis in cancer diagnosis and treatment.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of the protein of the present invention (1)
Is.

FIG. 2 shows the nucleotide sequence of the protein of the present invention (2)
Is.

FIG. 3 shows the nucleotide sequence of the protein of the present invention (3)
Is.

FIG. 4 shows the nucleotide sequence of the protein of the present invention (4)
Is.

FIG. 5 shows the nucleotide sequence of the protein of the present invention (5)
Is.

FIG. 6 shows the nucleotide sequence of the protein of the present invention (6)
Is.

FIG. 7 shows the nucleotide sequence of the protein of the present invention (7).
Is.

FIG. 8 is an electrophoretic image of a PCR product amplified using GSP1 and GSP4 as primers.

FIG. 9: Hepatoma cell line and non-cancer hepatocyte line (GAP
It is an image showing the expression levels of clone 141 and GAPDH in DH).

FIG. 10 is a graph showing the mRNA expression level of clone 141 standardized with GAPDH in each sample adjusted in the adjustment example.

FIG. 11 is a table showing the RNA expression state of the protein of the present invention in hepatocellular carcinoma tissues.

FIG. 12 is a graph showing the average expression level of mRNA of the protein of the present invention in liver cancer tissue (T) and non-cancer liver tissue (N).

FIG. 13 is a diagram showing mutation analysis of exon 3 of β-catenin gene, which is the 31st codon (TC) of Sample Example 24.
It is an electropherogram which shows the mutation of T to TGT.

FIG. 14 is a schematic diagram showing amino substitution in β-catenin mutation occurring in 16 cases out of 18 cases in which Gpr49 was overexpressed.

FIG. 15: MOCK, wild type β-catenin and mutant β-
Mouse MH infected with catenin by retrovirus
It is a graph which shows the expression level of FEX mRNA in T cell.

FIG. 16: Gpr of 38 cases of hepatocellular carcinoma obtained in the adjusted example
It is a table which shows the expression level of 49.

[Sequence list]                                SEQUENCE LISTING        <110> National Cancer Center Research Institute <120> Novel protein upragulated in cancer and gene encoding       thereof <130> 01XA019 <140> <141> <160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 4355 <212> DNA <213> Homo Sapiens <400> 1 tgctgctctc cgcccgcgtc cggctcgtgg ccccctactt cgggcaccat ggacacctcc 60 cggctcggtg tgctcctgtc cttgcctgtg ctgctgcagc tggcgaccgg gggcagctct 120 cccaggtctg gtgtgttgct gaggggctgc cccacacact gtcattgcga gcccgacggc 180 aggatgttgc tcagggtgga ctgctccgac ctggggctct cggagctgcc ttccaacctc 240 agcgtcttca cctcctacct agacctcagt atgaacaaca tcagtcagct gctcccgaat 300 cccctgccca gtctccgctt cctggaggag ttacgtcttg cgggaaacgc tctgacatac 360 attcccaagg gagcattcac tggcctttac agtcttaaag ttcttatgct gcagaataat 420 cagctaagac acgtacccac agaagctctg cagaatttgc gaagccttca atccctgcgt 480 ctggatgcta accacatcag ctatgtgccc ccaagctgtt tcagtggcct gcattccctg 540 aggcacctgt ggctggatga caatgcgtta acagaaatcc ccgtccaggc ttttagaagt 600 ttatcggcat tgcaagccat gaccttggcc ctgaacaaaa tacaccacat accagactat 660 gcctttggaa acctctccag cttggtagtt ctacatctcc ataacaatag aatccactcc 720 ctgggaaaga aatgctttga tgggctccac agcctagaga ctttagattt aaattacaat 780 aaccttgatg aattccccac tgcaattagg acactctcca accttaaaga actaggattt 840 catagcaaca atatcaggtc gatacctgag aaagcatttg taggcaaccc ttctcttatt 900 acaatacatt tctatgacaa tcccatccaa tttgttggga gatctgcttt tcaacattta 960 cctgaactaa gaacactgac tctgaatggt gcctcacaaa taactgaatt tcctgattta 1020 actggaactg caaacctgga gagtctgact ttaactggag cacagatctc atctcttcct 1080 caaaccgtct gcaatcagtt acctaatctc caagtgctag atctgtctta caacctatta 1140 gaagatttac ccagtttttc agtctgccaa aagcttcaga aaattgacct aagacataat 1200 gaaatctacg aaattaaagt tgacactttc cagcagttgc ttagcctccg atcgctgaat 1260 ttggcttgga acaaaattgc tattattcac cccaatgcat tttccacttt gccatcccta 1320 ataaagctgg acctatcgtc caacctcctg tcgtcttttc ctataactgg gttacatggt 1380 ttaactcact taaaattaac aggaaatcat gccttacaga gcttgatatc atctgaaaac 1440 tttccagaac tcaaggttat agaaatgcct tatgcttacc agtgctgtgc atttggagtg 1500 tgtgagaatg cctataagat ttctaatcaa tggaataaag gtgacaacag cagtatggac 1560 gaccttcata agaaagatgc tggaatgttt caggctcaag atgaacgtga ccttgaagat 1620 ttcctgcttg actttgagga agacctgaaa gcccttcatt cagtgcagtg ttcaccttcc 1680 ccaggcccct tcaaaccctg tgaacacctg cttgatggct ggctgatcag aattggagtg 1740 tggaccatag cagttctggc acttacttgt aatgctttgg tgacttcaac agttttcaga 1800 tcccctctgt acatttcccc cattaaactg ttaattgggg tcatcgcagc agtgaacatg 1860 ctcacgggag tctccagtgc cgtgctggct ggtgtggatg cgttcacttt tggcagcttt 1920 gcacgacatg gtgcctggtg ggagaatggg gttggttgcc atgtcattgg ttttttgtcc 1980 atttttgctt cagaatcatc tgttttcctg cttactctgg cagccctgga gcgtgggttc 2040 tctgtgaaat attctgcaaa atttgaaacg aaagctccat tttctagcct gaaagtaatc 2100 attttgctct gtgccctgct ggccttgacc atggccgcag ttcccctgct gggtggcagc 2160 aagtatggcg cctcccctct ctgcctgcct ttgccttttg gggagcccag caccatgggc 2220 tacatggtcg ctctcatctt gctcaattcc ctttgcttcc tcatgatgac cattgcctac 2280 accaagctct actgcaattt ggacaaggga gacctggaga atatttggga ctgctctatg 2340 gtaaaacaca ttgccctgtt gctcttcacc aactgcatcc taaactgccc tgtggctttc 2400 ttgtccttct cctctttaat aaaccttaca tttatcagtc ctgaagtaat taagtttatc 2460 cttctggtgg tagtcccact tcctgcatgt ctcaatcccc ttctctacat cttgttcaat 2520 cctcacttta aggaggatct ggtgagcctg agaaagcaaa cctacgtctg gacaagatca 2580 aaacacccaa gcttgatgtc aattaactct gatgatgtcg aaaaacagtc ctgtgactca 2640 actcaagcct tggtaacctt taccagctcc agcatcactt atgacctgcc tcccagttcc 2700 gtgccatcac cagcttatcc agtgactgag agctgccatc tttcctctgt ggcatttgtc 2760 ccatgtctct aattaatatg tgaaggaaaa tgttttcaaa ggttgagaac ctgaaaatgt 2820 gagattgagt atatcagagc agtaattaat aagaagagct gaggtgaaac tcggtttaaa 2880 aaccaaaaaa gaatctctca gttagtaaga aaaggctgaa aacctcttga tacttgagag 2940 tgaatataag tctaaatgct gctttgtata atttgttcag ctaagagata gatcggatca 3000 cactatttaa gtgagcccag atcaaaaaag cagattgaaa ttttctttag aaaagattct 3060 ccatgatttg aattgcattc tctttaaact caccaatgta atcattttgg gaggagggag 3120 aacccacttg ctttccaaat gggtttattt aaacccacaa actcaagagg ttgttggggg 3180 aattaggaaa ataagggttt tcaatgacct acattgctag gtagaggctg tgatccatgg 3240 gatttcattc taatgaccat gtgaagatgt ttgagtcctc ctttgccttt cctcagaaag 3300 aatccttcta aggcacaaat cccttagatg gataatgtaa ggtattgtta actcactcat 3360 attgagatca tttttagaga taccaggttt tatgtatcag cactagatgg ttccaccctc 3420 atgggataaa actgcttaca agtattttga aagaaaaact gaccaaaatt cttaaattgt 3480 tactaaggca atcatgcaca ggtgacgtat gtcttatctg atttgttttt aactccttgg 3540 tgcccaaagc tcagaaggga attccactgc cagcaatgaa catacctgga aaagaaagta 3600 agcaatctgg gatttttttt ctgggttagt aaagaatttt tgcaataagt tttatcagtt 3660 gattcaaact gatgtgcatc ttaatgatca aatgtgcaca ttacataaat taagtccact 3720 gatacaactt cttacacatg tatctctagt agctctggca aacccaatat ctgacaccac 3780 tttggactca agagactcag taacgtatta tcctgtttat ttagcttggt tttagctgtg 3840 ttctctctgg ataacccact tgatgttagg aacattactt ctctgcttat tccatattaa 3900 tactgtgtta ggtattttaa gaagcaagtt attaaataag aaaagtcaaa gtattaattc 3960 ttaccttcta ttatcctata ttagcttcaa tacatccaaa ccaaatggct gttaggtaga 4020 tttattttta tataagcatg tttattttga tcagatgttt taacttggat ttgaaaaaat 4080 acatttatga gatgttttat aagatgtgta aatatagaac tgtatttatt actatagtaa 4140 aggttcagta acattaagga ccatgataat gataataaac cttgtacagt ggcatattct 4200 ttgatttata ttgtgtttct ctgcccattt tctttaaatt cattaactgt atatatgtaa 4260 atatatagta cttgtaaata gattccnaat ttgcttttct attgggtaaa aaataaattt 4320 gtaataaaat gtgtgactat gaaaaaaaaa aaaaa 4355 <210> 2 <211> 907 <212> PRT <213> Homo Sapiens <400> 2 Met Asp Thr Ser Arg Leu Gly Val Leu Leu Ser Leu Pro Val Leu Leu   1 5 10 15 Gln Leu Ala Thr Gly Gly Ser Ser Pro Arg Ser Gly Val Leu Leu Arg              20 25 30 Gly Cys Pro Thr His Cys His Cys Glu Pro Asp Gly Arg Met Leu Leu          35 40 45 Arg Val Asp Cys Ser Asp Leu Gly Leu Ser Glu Leu Pro Ser Asn Leu      50 55 60 Ser Val Phe Thr Ser Tyr Leu Asp Leu Ser Met Asn Asn Ile Ser Gln  65 70 75 80 Leu Leu Pro Asn Pro Leu Pro Ser Leu Arg Phe Leu Glu Glu Leu Arg                  85 90 95 Leu Ala Gly Asn Ala Leu Thr Tyr Ile Pro Lys Gly Ala Phe Thr Gly             100 105 110 Leu Tyr Ser Leu Lys Val Leu Met Leu Gln Asn Asn Gln Leu Arg His         115 120 125 Val Pro Thr Glu Ala Leu Gln Asn Leu Arg Ser Leu Gln Ser Leu Arg     130 135 140 Leu Asp Ala Asn His Ile Ser Tyr Val Pro Pro Ser Cys Phe Ser Gly 145 150 155 160 Leu His Ser Leu Arg His Leu Trp Leu Asp Asp Asn Ala Leu Thr Glu                 165 170 175 Ile Pro Val Gln Ala Phe Arg Ser Leu Ser Ala Leu Gln Ala Met Thr             180 185 190 Leu Ala Leu Asn Lys Ile His His Ile Pro Asp Tyr Ala Phe Gly Asn         195 200 205 Leu Ser Ser Leu Val Val Leu His Leu His Asn Asn Arg Ile His Ser     210 215 220 Leu Gly Lys Lys Cys Phe Asp Gly Leu His Ser Leu Glu Thr Leu Asp 225 230 235 240 Leu Asn Tyr Asn Asn Leu Asp Glu Phe Pro Thr Ala Ile Arg Thr Leu                 245 250 255 Ser Asn Leu Lys Glu Leu Gly Phe His Ser Asn Asn Ile Arg Ser Ile             260 265 270 Pro Glu Lys Ala Phe Val Gly Asn Pro Ser Leu Ile Thr Ile His Phe         275 280 285 Tyr Asp Asn Pro Ile Gln Phe Val Gly Arg Ser Ala Phe Gln His Leu     290 295 300 Pro Glu Leu Arg Thr Leu Thr Leu Asn Gly Ala Ser Gln Ile Thr Glu 305 310 315 320 Phe Pro Asp Leu Thr Gly Thr Ala Asn Leu Glu Ser Leu Thr Leu Thr                 325 330 335 Gly Ala Gln Ile Ser Ser Leu Pro Gln Thr Val Cys Asn Gln Leu Pro             340 345 350 Asn Leu Gln Val Leu Asp Leu Ser Tyr Asn Leu Leu Glu Asp Leu Pro         355 360 365 Ser Phe Ser Val Cys Gln Lys Leu Gln Lys Ile Asp Leu Arg His Asn     370 375 380 Glu Ile Tyr Glu Ile Lys Val Asp Thr Phe Gln Gln Leu Leu Ser Leu 385 390 395 400 Arg Ser Leu Asn Leu Ala Trp Asn Lys Ile Ala Ile Ile His Pro Asn                 405 410 415 Ala Phe Ser Thr Leu Pro Ser Leu Ile Lys Leu Asp Leu Ser Ser Asn             420 425 430 Leu Leu Ser Ser Phe Pro Ile Thr Gly Leu His Gly Leu Thr His Leu         435 440 445 Lys Leu Thr Gly Asn His Ala Leu Gln Ser Leu Ile Ser Ser Glu Asn     450 455 460 Phe Pro Glu Leu Lys Val Ile Glu Met Pro Tyr Ala Tyr Gln Cys Cys 465 470 475 480 Ala Phe Gly Val Cys Glu Asn Ala Tyr Lys Ile Ser Asn Gln Trp Asn                 485 490 495 Lys Gly Asp Asn Ser Ser Met Asp Asp Leu His Lys Lys Asp Ala Gly             500 505 510 Met Phe Gln Ala Gln Asp Glu Arg Asp Leu Glu Asp Phe Leu Leu Asp         515 520 525 Phe Glu Glu Asp Leu Lys Ala Leu His Ser Val Gln Cys Ser Pro Ser     530 535 540 Pro Gly Pro Phe Lys Pro Cys Glu His Leu Leu Asp Gly Trp Leu Ile 545 550 555 560 Arg Ile Gly Val Trp Thr Ile Ala Val Leu Ala Leu Thr Cys Asn Ala                 565 570 575 Leu Val Thr Ser Thr Val Phe Arg Ser Pro Leu Tyr Ile Ser Pro Ile             580 585 590 Lys Leu Leu Ile Gly Val Ile Ala Ala Val Asn Met Leu Thr Gly Val         595 600 605 Ser Ser Ala Val Leu Ala Gly Val Asp Ala Phe Thr Phe Gly Ser Phe     610 615 620 Ala Arg His Gly Ala Trp Trp Glu Asn Gly Val Gly Cys His Val Ile 625 630 635 640 Gly Phe Leu Ser Ile Phe Ala Ser Glu Ser Ser Val Phe Leu Leu Thr                 645 650 655 Leu Ala Ala Leu Glu Arg Gly Phe Ser Val Lys Tyr Ser Ala Lys Phe             660 665 670 Glu Thr Lys Ala Pro Phe Ser Ser Leu Lys Val Ile Ile Leu Leu Cys         675 680 685 Ala Leu Leu Ala Leu Thr Met Ala Ala Val Pro Leu Leu Gly Gly Ser     690 695 700 Lys Tyr Gly Ala Ser Pro Leu Cys Leu Pro Leu Pro Phe Gly Glu Pro 705 710 715 720 Ser Thr Met Gly Tyr Met Val Ala Leu Ile Leu Leu Asn Ser Leu Cys                 725 730 735 Phe Leu Met Met Thr Ile Ala Tyr Thr Lys Leu Tyr Cys Asn Leu Asp             740 745 750 Lys Gly Asp Leu Glu Asn Ile Trp Asp Cys Ser Met Val Lys His Ile         755 760 765 Ala Leu Leu Leu Phe Thr Asn Cys Ile Leu Asn Cys Pro Val Ala Phe     770 775 780 Leu Ser Phe Ser Ser Leu Ile Asn Leu Thr Phe Ile Ser Pro Glu Val 785 790 795 800 Ile Lys Phe Ile Leu Leu Val Val Val Pro Leu Pro Ala Cys Leu Asn                 805 810 815 Pro Leu Leu Tyr Ile Leu Phe Asn Pro His Phe Lys Glu Asp Leu Val             820 825 830 Ser Leu Arg Lys Gln Thr Tyr Val Trp Thr Arg Ser Lys His Pro Ser         835 840 845 Leu Met Ser Ile Asn Ser Asp Asp Val Glu Lys Gln Ser Cys Asp Ser     850 855 860 Thr Gln Ala Leu Val Thr Phe Thr Ser Ser Ser Ile Thr Tyr Asp Leu 865 870 875 880 Pro Pro Ser Ser Val Pro Ser Pro Ala Tyr Pro Val Thr Glu Ser Cys                 885 890 895 His Leu Ser Ser Val Ala Phe Val Pro Cys Leu             900 905 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ctcgtggccc cctacttc 18 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gaggatctgg tgagcctgag aa 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cataagtgat gctggagctg gtaa 24 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 tcatggtcct taatgttact gaacc 25 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gagtcaaccc aagccttagt atcc 24 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 catgggacaa atgcaactga ag 22

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 33/53 D G01N 33/53 33/574 A 33/574 C12N 15/00 ZNAA (72 ) Inventor Norio Hirohashi 5-1-1, Tsukiji, Chuo-ku, Tokyo F-term in the National Cancer Center (reference) 4B024 AA01 AA11 BA63 CA04 GA11 HA14 4B063 QA01 QA13 QA19 QQ42 QR08 QR35 QR62 QR77 QS25 QS34 QX07 4C084 AA17 NA14 Z14 4H045 AA10 AA30 BA10 CA41 DA50 EA20 EA50 FA74

Claims (14)

[Claims] 1. An amino acid sequence of SEQ ID NO: 2 or one or several amino acids of the amino acid sequence of SEQ ID NO: 2 are added,
A protein containing a deleted or substituted amino acid sequence and being overexpressed in cancer cells. 2. The protein according to claim 1, wherein overexpression is induced by β-catenin mutation. 3. The protein according to claim 1, which is a G protein-coupled receptor and has SH2 and SH3 domains at the C-terminus. 4. The protein according to claim 1, wherein transcription is enhanced by the activity of the Wnt signal transduction system. 5. A base sequence of SEQ ID NO: 1 or a base sequence in which one or several bases of the base sequence of SEQ ID NO: 1 are added, deleted or substituted and its complementary sequence,
A protein comprising an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids of the amino acid sequence of SEQ ID NO: 2 are added, deleted or substituted, and which is overexpressed in cancer cells Gene to do. 6. One or several amino acids of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 is added,
A cancer diagnostic marker, comprising a protein containing a deleted or substituted amino acid sequence and showing overexpression in cancer cells. 7. A method for detecting cancer using a tissue collected from the inside of a subject, wherein one or several amino acids of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 is added or deleted. Alternatively, a method for detecting cancer, which comprises detecting a cancer using a diagnostic marker that includes a protein that has a substituted amino acid sequence and that is overexpressed in cancer cells. 8. A method for diagnosing cancer using cells / tissues collected from a subject, wherein one or a number of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 is contained in the cells / tissues. Of a cancer characterized by screening a protein containing an amino acid sequence in which individual amino acids are added, deleted or substituted, and showing overexpression in cancer cells, and diagnosing based on the expression of the protein Diagnostic method. 9. An amino acid sequence of SEQ ID NO: 2 expressed in a cancer cell tissue of a subject or an amino acid sequence in which one or several amino acids of the amino acid sequence of SEQ ID NO: 2 are added, deleted or substituted. A method for treating cancer, which comprises using a compound that specifically binds to a protein that is overexpressed in cancer cells, as a target. 10. A method for prognosis after cancer treatment, comprising removing a tissue of a cancer treatment site from within a subject, the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 in the molecule.
Characterized in that it screens for a protein containing an amino acid sequence in which one or several amino acids are added, deleted or substituted, and which is overexpressed in cancer cells, and makes a diagnosis based on the expression of the protein. Cancer prognosis method. 11. A method for treating cancer, comprising directly or indirectly comprising a plurality of genes including a nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof and a gene consisting of a part or all of these sequences and a gene product thereof. A method for treating cancer, which comprises administering an inhibitory compound that specifically binds to the gene or the gene product as a specific target. 12. A base sequence consisting of at least two of the base sequences of SEQ ID NO: 3 to SEQ ID NO: 8, wherein one or several bases of the base sequence of SEQ ID NO: 1 or the base sequence of SEQ ID NO: 1 is added,
A polynucleotide comprising a deleted or substituted base sequence and its complementary sequence, and used for the synthesis, amplification and detection of a gene and a protein which are overexpressed in cancer cells. 13. A polynucleotide comprising all or part of the polynucleotides of SEQ ID NO: 3 to SEQ ID NO: 8, wherein one or several amino acids of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 is added, deleted or A protein quantification kit, which comprises quantifying a protein containing a substituted amino acid sequence and showing overexpression in cancer cells. 14. A cancer cell screening kit comprising a plurality of gene fragments containing the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence and a gene consisting of part or all of these sequences.