JP2018191568A - Markers for detecting colon cancer - Google Patents

Abstract

To provide markers capable of rapid and accurate detection of colon cancer and pharmaceutical compositions for treating and/or preventing colon cancer.SOLUTION: The invention relates to a colon cancer detecting marker comprising a fusion gene or a polypeptide encoded by the fusion gene. The invention also relates to a method for assisting determination of the presence or absence or possibility of colon cancer affection including detection of the colon cancer detecting marker. The invention also relates to a pharmaceutical composition comprising an expression inhibitor of the fusion gene or a kinase inhibitor for treating and/or preventing colon cancer.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a marker for detecting colorectal cancer, a means for detecting the marker for detecting colorectal cancer, a method for assisting in the determination of the presence or absence of colorectal cancer, or the possibility of colorectal cancer, and Or it relates to a pharmaceutical composition for prevention.

  The large intestine is a digestive tract having a length of about 1.6 m in humans, and is roughly divided into three sites: the cecum, the rectum, and the colon. The large intestine is an important organ responsible for functions such as fermentation of dietary fiber by bacteria and absorption of moisture. In Japan, the number of patients with colorectal cancer has been increasing in recent years. As of 2014, it is estimated that the number of patients with colorectal cancer is approximately 250,000 or more, and the number of deaths is large. Detection methods and effective treatment methods are eagerly desired.

  As one of the main examination methods for colorectal cancer, there is a fecal occult blood test for detecting blood mixed in stool resulting from a tumor. In this method, the presence or absence of blood present in stool is examined using a monoclonal antibody against hemoglobin present in blood. In addition, a method using a blood marker such as CEA and CA19-9 has been reported for detection of colorectal cancer (Non-patent Document 1). However, neither the fecal occult blood test nor the above-mentioned blood marker was sufficient in terms of the sensitivity and specificity of colorectal cancer detection.

  In addition, about 10% of colorectal cancers are classified as microsatellite instability (MSI-H) colorectal cancer in which a mutation occurs in a repetitive sequence of microsatellite. Microsatellite unstable colorectal cancer (MSI-H CRC) is associated with Lynch syndrome (LS), a hereditary disease that increases the risk of developing cancer, including colorectal cancer (Non-patent Document 2). Since drug sensitivity and the like are different from those of other colorectal cancers, there is a need for a method capable of rapid detection. Normally, MSI can be detected by microsatellite markers (BAT25, BAT26, D2S123, D5S346, D17S250, etc.) using the PCR method (Non-patent Document 3), but this method is sufficient in terms of rapidity. It was not something that could be said.

Sisik A. et al., Asian Pac. J. Cancer Prev., 2013, 14 (7), pp. 4289-94. Lynch HT. Et al., Nat. Rev. Cancer, 2015, pp. 181-194. Boland CR. Et al., Cancer Res., 1998, 58, pp. 5248-5257.

  An object of the present invention is to provide a marker capable of quickly and accurately detecting colorectal cancer. Another object of the present invention is to provide a pharmaceutical composition for treating and / or preventing colorectal cancer.

The present inventor has found a novel fusion gene that can be used for detection of colorectal cancer. Moreover, when a kinase inhibitor was added with respect to the cell which has the said fusion gene, it discovered that excessive cell growth was suppressed and completed this invention. Accordingly, the present invention includes the following aspects.
(1) The following (i) to (v):
(I) a fusion gene of any gene and INSR gene,
(Ii) RUFY1 gene or NCOA4 gene and RET gene fusion gene,
(Iii) KANK1 gene or EML4 gene and NTRK3 gene fusion gene,
(Iv) ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, and (v) a fusion gene of LMNA gene or TPM3 gene and NTRK1 gene,
A marker for detecting colorectal cancer comprising a fusion gene selected from the group consisting of: or a polypeptide encoded by the fusion gene.
(2) The INSR gene, RUFY1 gene, NCOA4 gene, RET gene, KANK1 gene, EML4 gene, NTRK3 gene, AMRC10 gene, AKAP9 gene, AGAP3 gene, TRIM24 gene, BRAF gene, LMNA gene, TPM3 gene, and NTRK1 gene , Encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, respectively (1) Marker described in.
(3) The marker according to (1) or (2), wherein the arbitrary gene described in (i) is an SLC12A2 gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32.
(4) The fusion gene according to (i) encodes a polypeptide containing a kinase domain derived from the INSR gene,
The fusion gene described in (ii) encodes a polypeptide comprising a coiled coil domain of the RUFY1 gene or a part thereof or a coiled coil domain derived from the NCOA4 gene, and a kinase domain derived from the RET gene,
The fusion gene according to (iii) encodes a polypeptide comprising a coiled coil domain derived from the KANK1 gene or EML4 gene, and a kinase domain derived from the NTRK3 gene,
(Iv) the fusion gene described in (a) ARMC10 gene-derived ARM domain, AKAP9 gene-derived coiled-coil domain, AGAP3 gene-derived PH domain, or TRIM24 gene-derived RING domain, and (b) BRAF gene-derived Encodes a polypeptide comprising a kinase domain,
(V) The marker according to any one of (1) to (3), which encodes a polypeptide comprising a part of a coiled-coil domain derived from the LMNA gene or TPM3 gene and a kinase domain derived from the NTRK1 gene.
(5) In (i), the kinase domain derived from the INSR gene comprises the amino acid sequence of positions 1011 to 1286 of SEQ ID NO: 2,
In (ii), the coiled coil domain derived from the RUFY1 gene or a part thereof includes the amino acid sequences of positions 321 to 374 and / or 405 to 553 of SEQ ID NO: 4, and the coiled coil domain derived from the NCOA4 gene is represented by 1 to 2 of SEQ ID NO: 6. Comprising the amino acid sequence at position 200, the kinase domain derived from the RET gene comprising the amino acid sequence at positions 724 to 1016 of SEQ ID NO: 8,
In (iii), the coiled coil domain derived from the KANK1 gene contains at least one of the amino acid sequences of positions 258 to 316, 361 to 395, and 446 to 500 of SEQ ID NO: 10, and the coiled coil domain derived from the EML4 gene is SEQ ID NO: 12 comprising the amino acid sequence of positions 17 to 51, the kinase domain derived from the NTRK3 gene comprises the amino acid sequence of positions 538 to 825 of SEQ ID NO: 14,
In (iv), the ARM domain derived from the ARMC10 gene contains the amino acid sequence of positions 55 to 140 of SEQ ID NO: 16, the coiled coil domain derived from the AKAP9 gene is the amino acid sequence of positions 164 to 914 of SEQ ID NO: 18, and positions 944 to 1022 Amino acid sequence, amino acid sequence from 1100 to 1185, amino acid sequence from 1253 to 1280, amino acid sequence from 1336 to 1392, amino acid sequence from 1434 to 1459, amino acid sequence from 1585 to 1659, amino acid sequence from 1857 to 2455 AGAP3 gene comprising at least one of amino acid sequences from 2544 to 2561, amino acid sequences from 2603 to 2776, amino acids from 3065 to 3092, amino acids from 3124 to 3470, and amino acids from 3587 to 3689 The derived PH domain includes the amino acid sequence of positions 404 to 465 of SEQ ID NO: 20, the RING domain derived from the TRIM24 gene includes the amino acid sequence of positions 56 to 82 of SEQ ID NO: 22, and the kinase domain derived from the BRAF gene includes SEQ ID NO: 24 Of 457-717 position amino acid sequence,
In (v), the LMNA gene-derived coiled-coil domain includes amino acid sequences of positions 34 to 218 and / or 243 to 383 of SEQ ID NO: 26, and a part of the coiled-coil domain derived from the TPM3 gene is 1-211 of SEQ ID NO: 28 The kinase domain derived from the NTRK1 gene comprises the amino acid sequence of positions 510 to 781 of SEQ ID NO: 30,
The marker according to (4).
(6) a fusion gene of an arbitrary gene and the INSR gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 34;
RUFY1 gene or NCOA4 gene and RET gene fusion gene each encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 36 or 38,
A fusion gene of KANK1 gene or EML4 gene and NTRK3 gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 40 or 42, respectively;
ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene each encodes a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 44, 46, 48, or 50,
The marker according to (5), wherein the LMNA gene or the fusion gene of TPM3 gene and NTRK1 gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 52 or 54, respectively.
(7) The marker according to any one of (1) to (6), wherein the colon cancer is microsatellite unstable colon cancer.
(8) A kit comprising a primer set, a probe, an aptamer, an antibody, or at least one of them for detecting the marker according to any one of (1) to (7).
(9) In the sample obtained from the subject, assisting the determination of the presence or absence of colorectal cancer or the possibility of colorectal cancer including the step of detecting the marker according to any of (1) to (7) how to.
(10) The following (i) to (v):
(I) a fusion gene of any gene and INSR gene,
(Ii) RUFY1 gene or NCOA4 gene and RET gene fusion gene,
(Iii) KANK1 gene or EML4 gene and NTRK3 gene fusion gene,
(Iv) ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, and (v) a fusion gene of LMNA gene or TPM3 gene and NTRK1 gene,
A pharmaceutical composition comprising a fusion gene expression inhibitor or a kinase inhibitor for treating colorectal cancer in a subject having at least one fusion gene selected from the group consisting of:
When the subject has the fusion gene described in (i), the kinase inhibitor is an INSR inhibitor;
If the subject has at least one of the fusion genes described in (ii), the kinase inhibitor is a RET inhibitor;
When the subject has at least one of the fusion genes described in (iii), the kinase inhibitor is an NTRK3 inhibitor;
If the subject has at least one of the fusion genes described in (iv), the kinase inhibitor is a BRAF inhibitor;
If the subject has at least one of the fusion genes described in (v), the kinase inhibitor is an NTRK1 inhibitor;
Said pharmaceutical composition.
(11) The INSR gene, RUFY1 gene, NCOA4 gene, RET gene, KANK1 gene, EML4 gene, NTRK3 gene, AMRC10 gene, AKAP9 gene, AGAP3 gene, TRIM24 gene, BRAF gene, LMNA gene, TPM3 gene, and NTRK1 gene , Encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, respectively (10) A pharmaceutical composition according to 1.
(12) The pharmaceutical composition according to (10) or (11), wherein the arbitrary gene described in (i) is the SLC12A2 gene, and the SLC12A2 gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32 .
(13) The fusion gene expression inhibitor is an RNAi molecule, or the kinase inhibitor is at least one drug selected from the group consisting of aptamers, neutralizing antibodies, and low molecular weight compounds. )-(12) The pharmaceutical composition in any one of.
(14) the amino acid sequence represented by any of SEQ ID NOs: 34, 36, and 46,
An amino acid sequence having 90% or more identity to the amino acid sequence represented by any one of SEQ ID NOs: 34, 36, and 46, or 1 or A polypeptide comprising an amino acid sequence in which several amino acids have been added, deleted and / or substituted.
(15) A polynucleotide encoding the polypeptide according to (14).

  According to the present invention, a marker and the like capable of detecting colorectal cancer quickly and accurately are provided. In addition, the present invention provides a pharmaceutical composition and the like for effectively treating and / or preventing colorectal cancer.

FIG. 1 shows a schematic diagram of a polypeptide encoded by a fusion gene found in the Examples herein. In the fusion polypeptide, the N-terminal polypeptide is shown in white, the C-terminal polypeptide is shown in dots, and the domains in each polypeptide are shown in diagonal lines. In the figure, SLC12A2-INSR indicates a polypeptide encoded by a fusion gene of SLC12A2 gene and INSR gene. RUFY1-RET and NCOA4-RET indicate polypeptides encoded by the RUFY1 gene or a fusion gene of NCOA4 and RET gene, respectively. LMNA-NTRK1 and TPM3-NTRK1 represent polypeptides encoded by the LMNA gene or a fusion gene of TPM3 gene and NTRK1 gene, respectively. EML4-NTRK3 and KANK1-NTRK3 represent polypeptides encoded by the EML4 gene or a fusion gene of KANK1 gene and NTRK3 gene, respectively. AMRC10-BRAF, AGAP3-BRAF, TRIM24-BRAF, and AKAP9-BRAF are polypeptides encoded by AMRC10 gene, AGAP3 gene, TRIM24 gene, or a fusion gene of AKAP9 gene and BRAF gene, respectively. The numbers shown below the polypeptides refer to the amino acid positions in the wild-type polypeptide of each polypeptide that makes up the fusion polypeptide. CC refers to the coiled-coil domain, ARM refers to the ARM domain, PH refers to the PH domain, and kinase refers to the protein kinase domain. Some polypeptides encoded by the fusion gene have a plurality of CC domains, but these are shown as one CC domain for convenience. FIG. 2 is a diagram showing the results of 3T3 transformation assay of the fusion polypeptides found in the examples. The notation of the fusion gene is based on Fig. 1. FIG. 3 is a graph showing the cell density when a kinase inhibitor is added to cells in which the fusion polypeptide found in the Examples is expressed. The notation of the fusion gene is based on Fig. 1. By adding an agent that inhibits the kinase in each fusion polypeptide, the cell density of 3T3 cells expressing the fusion polypeptide was reduced in a concentration-dependent manner.

<Colorectal cancer detection marker>
In one aspect, the present invention relates to a marker for detecting colorectal cancer. “Colorectal cancer detection marker” means a biomarker that serves as an index for detecting colorectal cancer. By detecting the presence of a marker for detecting colorectal cancer in a sample derived from a subject, it is possible to determine the presence or absence of colorectal cancer injury or the possibility of colorectal cancer in the subject. In the present specification, the species of the “subject” is not limited, but is preferably a mammal, for example, a primate such as a human and a chimpanzee, a laboratory animal such as a rat and a mouse, a pig, a cow, a horse, a sheep, and a goat. Domestic animals such as dogs, pets such as dogs and cats, preferably humans.

  The colorectal cancer that can be detected by the marker of the present invention may be any of cecal cancer, colon cancer, and rectal cancer. The colorectal cancer may also be microsatellite instability (MSI-H) colorectal cancer. In this specification, “microsatellite instability-high colorectal cancer (MSI-CRC)” is mutated to the length of a repeat unit (microsatellite) of 1 to 5 bases on the genome (microsatellite anxiety). It means colon cancer with qualitative (MSI) (Virginia Pinol, JAMA., 2005, 293 (16), pp. 1986-1994). In one embodiment, microsatellite instability is defined as a condition that exhibits MSI in BAT25 or BAT426, preferably both BAT25 and BAT426. BAT25 and BAT426 are mononucleotide repeats widely used as MSI markers. Microsatellite unstable colorectal cancer is a LS (Lynch syndrome) subtype that has a mutation in the MMR (mismatch repair) gene, and the MLH1 (mutL homolog 1) promoter has high methylation frequency (for example, , Β value> 0.2%) MM (MLH1 methylated) subtype, LL (Lynch-like) subtype that has no mutation in MMR gene, and MLH1 gene promoter and methylation frequency is low It can be further classified into types.

  The marker for colorectal cancer detection of the present invention comprises (i) a fusion gene of any gene and INSR gene, (ii) a fusion gene of RUFY1 gene or NCOA4 gene and RET gene, (iii) KANK1 gene or EML4 gene and NTRK3 gene A fusion gene selected from the group consisting of a fusion gene, (iv) ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, and (v) a fusion gene of LMNA gene or TPM3 gene and NTRK1 gene, Alternatively, it consists of a polypeptide encoded by the fusion gene. The fusion gene is preferably selected from the group consisting of the above (i) to (iii), more preferably the fusion gene described in (i).

  In principle, the fusion gene constituting the marker for detecting colorectal cancer of the present invention has a carcinogenic potential. Presence / absence of canceration can be identified by a method known in the art. For example, by introducing a fusion gene into an appropriate cell (for example, a mammalian cell) to proliferate and observing the presence or absence of focus formation, For example, it can be determined by observing loss of contact inhibition and / or acquisition of anchorage-independent growth ability. For details of the method for measuring the presence or absence of canceration ability, see, for example, the Examples of the present specification.

  The fusion gene constituting the marker for colorectal cancer detection of the present invention and each gene contained in the fusion gene will be described in detail below.

(I) Fusion gene of arbitrary gene and INSR gene In one embodiment, the marker for colorectal cancer detection of the present invention is a fusion gene of an arbitrary gene and INSR (insulin receptor) gene, or a polycode encoded by the fusion gene. It consists of peptides. Here, the arbitrary gene is not limited as long as a fusion gene having canceration ability is produced by fusion with the INSR gene, but is preferably an SLC12A2 (Solute carrier family 12 member 2) gene.

  In the present specification, the SLC12A2 gene may preferably be a polynucleotide comprising the base sequence of the human SLC12A2 gene represented by SEQ ID NO: 31 or a functionally equivalent base sequence thereof. Further, the polypeptide encoded by the SLC12A2 gene may be a polypeptide comprising the amino acid sequence of the human SLC12A2 protein represented by SEQ ID NO: 32 or a functionally equivalent amino acid sequence thereof.

  In this specification, a certain base sequence and a “functionally equivalent base sequence” refer to a base sequence having a biological function equivalent to the original base sequence. For example, for the original base sequence, For example, a base sequence having 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity, or one or more bases added in the original base sequence , Deleted and / or substituted nucleotide sequences. Similarly, a “functionally equivalent amino acid sequence” to an amino acid sequence refers to an amino acid sequence having a biological function equivalent to that of the original amino acid sequence. % Or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of an amino acid sequence having one or more amino acids added or missing in the original amino acid sequence It may be a missing and / or substituted amino acid sequence.

  In the present specification, the identity value relating to the base sequence and amino acid sequence is a value calculated with default settings using software (for example, FASTA, DANASYS, and BLAST) that calculates identity between a plurality of sequences. . For details on how identity is determined, see, for example, Altschul et al., Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990. I want to be. In the present specification, the range of “one or more” is 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, or 1 or 2 It is a piece.

  In the present specification, the INSR gene may preferably be a polynucleotide comprising the base sequence of the human INSR gene represented by SEQ ID NO: 1 or a functionally equivalent base sequence thereof. Further, the polypeptide encoded by the INSR gene may be a polypeptide comprising the amino acid sequence of the human INSR protein represented by SEQ ID NO: 2 or a functionally equivalent amino acid sequence thereof.

  The fusion form of the fusion gene of an arbitrary gene and INSR gene is not limited as long as it has canceration ability, but includes a sequence on the N-terminal side of a polypeptide encoded by an arbitrary gene such as the SLC12A2 gene, Those encoding a polypeptide containing a C-terminal sequence of the polypeptide encoded by the INSR gene on the C-terminal side are preferred. Preferably, the fusion gene encodes a polypeptide comprising a kinase domain from the INSR gene (a polypeptide encoded by). The kinase domain derived from the INSR gene includes the amino acid sequence at positions 1011 to 1286 of SEQ ID NO: 2, or a functionally equivalent amino acid sequence thereof.

  The fusion gene of any gene and INSR gene is preferably a polynucleotide comprising the base sequence of the fusion gene of human SLC12A2 gene and human INSR gene represented by SEQ ID NO: 33, or a functionally equivalent base sequence thereof. Good. The polypeptide encoded by the fusion gene of any gene and the INSR gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 34, or a functionally equivalent amino acid sequence thereof.

  For example, the base sequence functionally equivalent to the base sequence represented by SEQ ID NO: 1, 31, or 33 is, for example, 80% or more, preferably the base sequence represented by SEQ ID NO: 1, 31, or 33, respectively. 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the nucleotide sequence, or one or more bases in the nucleotide sequence represented by SEQ ID NO: 1, 31, or 33 The nucleotide sequence may be added, deleted, and / or substituted. In addition, the amino acid sequence functionally equivalent to the amino acid sequence represented by SEQ ID NO: 2, 32, or 34 is, for example, 80% or more with respect to the amino acid sequence represented by SEQ ID NO: 2, 32, or 34, preferably An amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity, or one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2, 32, or 34 It may be an amino acid sequence that has been added, deleted, and / or substituted. Similarly, the amino acid sequence functionally equivalent to the amino acid sequence of positions 1011 to 1286 of SEQ ID NO: 2 is, for example, 80% or more, preferably 90% or more, relative to the amino acid sequence of positions 1011 to 1286 of SEQ ID NO: 2. An amino acid sequence having 95% or more, 97% or more, 98% or more, or 99% or more identity, or one or more amino acids added or deleted in the amino acid sequence of positions 1011 to 1286 of SEQ ID NO: 2, and It may be a substituted amino acid sequence.

(Ii) Fusion gene of RUFY1 gene or NCOA4 gene and RET gene In one embodiment, the marker for colorectal cancer detection of the present invention comprises a RUFY1 (RUN and FYVE domain containing 1) gene and a RET (or RET proto-oncogene) gene. It consists of a fusion gene, a fusion gene of NCOA4 (nuclear receptor coactivator 4) gene and RET gene, or a polypeptide encoded by the fusion gene.

  In the present specification, the RUFY1 gene may preferably be a polynucleotide comprising the base sequence of the human RUFY1 gene represented by SEQ ID NO: 3 or a functionally equivalent base sequence thereof. Further, the polypeptide encoded by the RUFY1 gene may be a polypeptide comprising the amino acid sequence of human SLC12A2 protein represented by SEQ ID NO: 4 or a functionally equivalent amino acid sequence thereof.

  In the present specification, the NCOA4 gene may preferably be a polynucleotide comprising the base sequence of the human NCOA4 gene represented by SEQ ID NO: 5 or a functionally equivalent base sequence thereof. The polypeptide encoded by the NCOA4 gene may be a polypeptide comprising the amino acid sequence of human NCOA4 protein represented by SEQ ID NO: 6 or a functionally equivalent amino acid sequence thereof.

  In the present specification, the RET gene may preferably be a polynucleotide comprising the base sequence of the human RET gene represented by SEQ ID NO: 7 or a functionally equivalent base sequence thereof. Further, the polypeptide encoded by the RET gene may be a polypeptide comprising the amino acid sequence of human RET protein represented by SEQ ID NO: 8 or a functionally equivalent amino acid sequence thereof.

  The fusion form of the fusion gene of RUFY1 gene or NCOA4 gene and RET gene is not limited as long as it has canceration ability, but includes the sequence on the N-terminal side of the polypeptide encoded by RUFY1 gene or NCOA4 gene on the N-terminal side, Those encoding a polypeptide containing a C-terminal sequence of the polypeptide encoded by the RET gene on the C-terminal side are preferred.

  The fusion gene is preferably a coiled-coil domain derived from the polypeptide encoded by the RUFY1 gene or a part thereof, or a coiled-coil domain derived from the polypeptide encoded by the NCOA4 gene, and a kinase derived from the polypeptide encoded by the RET gene Encodes a polypeptide containing a domain. The coiled-coil domain derived from the RUFY1 gene or a part thereof includes, for example, at least one of amino acid sequences of positions 321 to 374 and amino acids of positions 405 to 553 of SEQ ID NO: 4, or a functionally equivalent amino acid sequence thereof. The coiled coil domain derived from the NCOA4 gene includes, for example, the amino acid sequence at positions 1 to 200 of SEQ ID NO: 6, or a functionally equivalent amino acid sequence thereof. The kinase domain derived from the RET gene includes, for example, the amino acid sequence at positions 724 to 1016 of SEQ ID NO: 8, or a functionally equivalent amino acid sequence thereof.

  The fusion gene of RUFY1 gene and RET gene may preferably be a polynucleotide comprising the nucleotide sequence of the fusion gene of human RUFY1 gene and human RET gene represented by SEQ ID NO: 35, or a functionally equivalent nucleotide sequence thereof. . The polypeptide encoded by the fusion gene of the RUFY1 gene and the RET gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 36, or a functionally equivalent amino acid sequence thereof.

  The fusion gene of NCOA4 gene and RET gene is preferably a polynucleotide comprising the nucleotide sequence of the fusion gene of human NCOA4 gene and human RET gene represented by SEQ ID NO: 37, or a functionally equivalent nucleotide sequence thereof. It's okay. The polypeptide encoded by the fusion gene of NCOA4 gene and RET gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 38, or a functionally equivalent amino acid sequence thereof.

  For example, the base sequence functionally equivalent to the base sequence represented by SEQ ID NO: 3, 5, 7, 35, or 37 is, for example, the base sequence represented by SEQ ID NO: 3, 5, 7, 35, or 37, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or a nucleotide sequence having identity of 99% or more, or a base represented by SEQ ID NO: 3, 5, 7, 35 or 37 It may be a base sequence in which one or more bases are added, deleted, and / or substituted in the sequence. In addition, the amino acid sequence functionally equivalent to the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 36, or 38 is the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 36, or 38, respectively. An amino acid sequence having, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more, or SEQ ID NO: 4, 6, 8, 36, or 38 It may be an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown.

  Similarly, the amino acid sequence of positions 321 to 374 of SEQ ID NO: 4, the amino acid sequence of positions 405 to 553 of SEQ ID NO: 4, the amino acid sequence of positions 1 to 200 of SEQ ID NO: 6, or the amino acids of positions 724 to 1016 of SEQ ID NO: 8 The amino acid sequences functionally equivalent to the sequences are the amino acid sequences of positions 321 to 374 of SEQ ID NO: 4, the amino acid sequences of positions 405 to 553 of SEQ ID NO: 4, the amino acid sequences of positions 1 to 200 of SEQ ID NO: 6, or For example, an amino acid sequence having 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence at positions 724 to 1016 of SEQ ID NO: 8, Or the amino acid sequence of positions 321 to 374 of SEQ ID NO: 4, the amino acid sequence of positions 405 to 553 of SEQ ID NO: 4, the amino acid sequence of positions 1 to 200 of SEQ ID NO: 6, or the amino acid sequence of positions 724 to 1016 of SEQ ID NO: 8 Amino acid sequence in which one or more amino acids are added, deleted, and / or substituted It may be.

(Iii) KANK1 gene or fusion gene of EML4 gene and NTRK3 gene In one embodiment, the marker for colorectal cancer detection of the present invention comprises a KANK1 (KN motif and ankyrin repeat domains 1) gene and an NTRK3 (neurotrophic receptor tyrosine kinase 3) gene Or a fusion gene of EML4 (echinoderm microtubule associated protein like 4) gene and NTRK3 gene, or a polypeptide encoded by the fusion gene.

  In the present specification, the KANK1 gene may preferably be a polynucleotide comprising the base sequence of the human KANK1 gene represented by SEQ ID NO: 9, or a functionally equivalent base sequence thereof. The polypeptide encoded by the KANK1 gene may be a polypeptide comprising the amino acid sequence of the human KANK1 protein represented by SEQ ID NO: 10, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the EML4 gene may preferably be a polynucleotide comprising the base sequence of the human EML4 gene represented by SEQ ID NO: 11, or a functionally equivalent base sequence thereof. Further, the polypeptide encoded by the EML4 gene may be a polypeptide comprising the amino acid sequence of the human EML4 protein represented by SEQ ID NO: 12, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the NTRK3 gene is preferably 80% or more, preferably 90% or more, preferably 95% or more with respect to the nucleotide sequence of the human NTRK3 gene represented by SEQ ID NO: 13, and the nucleotide sequence represented by SEQ ID NO: 13. , 97% or more, 98% or more, or 99% or more of the nucleotide sequence, or a polynucleotide containing a functionally equivalent nucleotide sequence thereof. The polypeptide encoded by the NTRK3 gene may be a polypeptide comprising the amino acid sequence of human NTRK3 protein represented by SEQ ID NO: 14, or a functionally equivalent amino acid sequence thereof.

  The fusion form of the fusion gene is not limited as long as it has cancerigenic potential, but includes a sequence on the N-terminal side of the polypeptide encoded by the KANK1 gene or EML4 gene on the N-terminal side of the polypeptide encoded by the NTRK3 gene. Those encoding a polypeptide containing a C-terminal sequence on the C-terminal side are preferred.

  The fusion gene preferably encodes a polypeptide comprising a polypeptide encoded by the KANK1 gene or a coiled-coil domain derived from a polypeptide encoded by the EML4 gene and a kinase domain derived from the NTRK3 gene. The coiled coil domain derived from the KANK1 gene includes, for example, at least one of the amino acid sequences of positions 258 to 316, 361 to 395, and 446 to 500 of SEQ ID NO: 10, or a functionally equivalent amino acid sequence thereof. The coiled coil domain derived from the EML4 gene includes, for example, the amino acid sequence at positions 17 to 51 of SEQ ID NO: 12, or a functionally equivalent amino acid sequence thereof. In addition, the kinase domain derived from the NTRK3 gene includes, for example, the amino acid sequence at positions 538 to 825 of SEQ ID NO: 14, or a functionally equivalent amino acid sequence thereof.

  The fusion gene of KANK1 gene and NTRK3 gene may preferably be a polynucleotide comprising the base sequence of the fusion gene of human KANK1 gene and human NTRK3 gene represented by SEQ ID NO: 39, or a functionally equivalent base sequence thereof. . The polypeptide encoded by the fusion gene of KANK1 gene and NTRK3 gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 40 or a functionally equivalent amino acid sequence thereof.

  The fusion gene of EML4 gene and NTRK3 gene may preferably be a polynucleotide comprising the base sequence of the fusion gene of human EML4 gene and human NTRK3 gene represented by SEQ ID NO: 41, or a functionally equivalent base sequence thereof. . The polypeptide encoded by the fusion gene of EML4 gene and NTRK3 gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 42, or a functionally equivalent amino acid sequence thereof.

  For example, the base sequence functionally equivalent to the base sequence represented by SEQ ID NO: 9, 11, 13, 39 or 41 is, for example, the base sequence represented by SEQ ID NO: 9, 11, 13, 39 or 41, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or a nucleotide sequence having identity of 99% or more, or a base represented by SEQ ID NO: 9, 11, 13, 39 or 41 It may be a base sequence in which one or more bases are added, deleted, and / or substituted in the sequence. In addition, the amino acid sequence functionally equivalent to the amino acid sequence represented by SEQ ID NO: 10, 12, 14, 40, or 42 is the amino acid sequence represented by SEQ ID NO: 10, 12, 14, 40, or 42, respectively. An amino acid sequence having, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more, or SEQ ID NOs: 10, 12, 14, 40, or 42 It may be an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown.

  Similarly, the amino acid sequence of positions 258 to 316 of SEQ ID NO: 10, the amino acid sequence of positions 361 to 395 of SEQ ID NO: 10, the amino acid sequence of positions 446 to 500 of SEQ ID NO: 10, the amino acid sequence of positions 17 to 51 of SEQ ID NO: 12 Or the amino acid sequence functionally equivalent to the amino acid sequence of positions 538 to 825 of SEQ ID NO: 14, the amino acid sequence of positions 258 to 316 of SEQ ID NO: 10, the amino acid sequence of positions 361 to 395 of SEQ ID NO: 10, With respect to the amino acid sequence at positions 446 to 500 of No. 10, the amino acid sequence at positions 17 to 51 of SEQ ID NO: 12, or the amino acid sequence at positions 538 to 825 of SEQ ID NO: 14, for example, 80% or more, preferably 90% or more, Amino acid sequence having 95% or more, 97% or more, 98% or more, or 99% or more identity, or amino acid sequence of positions 258 to 316 of SEQ ID NO: 10, amino acid sequence of positions 361 to 395 of SEQ ID NO: 10, or sequence The amino acid sequence at positions 446 to 500 of No. 10, the amino acid sequence at positions 17 to 51 of SEQ ID No. 12, or the sequence It may be an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence at positions 538 to 825 of No. 14.

(Iv) ARMC10 gene, AKAP9 gene, AGAP3 gene, or fusion gene of TRIM24 gene and BRAF gene In one embodiment, the marker for colorectal cancer detection of the present invention is an ARMC10 (armadillo repeat containing 10) gene, AKAP9 (A-kinase anchoring) a gene selected from the group consisting of a protein 9) gene, an AGAP3 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 3) gene, and a TRIM24 (tripartite motif containing 24) gene; and BRAF (B-Raf proto-oncogene, serine / threonine kinase) gene or a polypeptide encoded by the fusion gene.

  In the present specification, the ARMC10 gene may preferably be a polynucleotide comprising the base sequence of the human ARMC10 gene represented by SEQ ID NO: 15, or a functionally equivalent base sequence thereof. The polypeptide encoded by the ARMC10 gene may be a polypeptide comprising the amino acid sequence of the human ARMC10 protein represented by SEQ ID NO: 16, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the AKAP9 gene may preferably be a polynucleotide comprising the base sequence of the human AKAP9 gene represented by SEQ ID NO: 17, or a functionally equivalent base sequence thereof. The polypeptide encoded by the AKAP9 gene may be a polypeptide comprising the amino acid sequence of the human AKAP9 protein represented by SEQ ID NO: 18, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the AGAP3 gene may preferably be a polynucleotide comprising the base sequence of the human AGAP3 gene represented by SEQ ID NO: 19, or a functionally equivalent base sequence thereof. The polypeptide encoded by the AGAP3 gene may be a polypeptide containing the amino acid sequence of the human AGAP3 protein represented by SEQ ID NO: 20, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the TRIM24 gene may preferably be a polynucleotide comprising the base sequence of the human TRIM24 gene represented by SEQ ID NO: 21, or a functionally equivalent base sequence thereof. The polypeptide encoded by the TRIM24 gene may be a polypeptide containing the amino acid sequence of human TRIM24 protein represented by SEQ ID NO: 22, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the BRAF gene may preferably be a polynucleotide comprising the base sequence of the human BRAF gene represented by SEQ ID NO: 23, or a functionally equivalent base sequence thereof. Further, the polypeptide encoded by the BRAF gene may be a polypeptide comprising the amino acid sequence of the human BRAF protein represented by SEQ ID NO: 24, or a functionally equivalent amino acid sequence thereof.

  The fusion form of the fusion gene is not limited as long as it has canceration ability, but the N-terminal sequence of a polypeptide encoded by a gene selected from the group consisting of ARMC10 gene, AKAP9 gene, AGAP3 gene, and TRIM24 gene It preferably encodes a polypeptide that is contained on the N-terminal side and contains a C-terminal sequence on the C-terminal side of the polypeptide encoded by the BRAF gene.

  The fusion gene is preferably (a) an ARM (Armadillo) domain derived from a polypeptide encoded by the ARMC10 gene, a coiled-coil domain derived from a polypeptide encoded by the AKAP9 gene, a PH derived from a polypeptide encoded by the AGAP3 gene ( A polypeptide comprising a Pleckstrin-homology) domain, or a RING (Really Interesting New Gene) domain derived from a polypeptide encoded by the TRIM24 gene, and (b) a kinase domain derived from a polypeptide encoded by the BRAF gene.

  The ARM domain derived from the ARMC10 gene includes, for example, the amino acid sequence at positions 55 to 140 of SEQ ID NO: 16, or a functionally equivalent amino acid sequence thereof. The coiled coil domain derived from the AKAP9 gene has, for example, the amino acid sequence at positions 164 to 914 of SEQ ID NO: 18, the amino acid sequence at positions 944 to 1022, the amino acid sequence at positions 1100 to 1185, the amino acid sequence at positions 1253 to 1280, and the amino acid sequence at positions 1336 to 1392 Amino acid sequence, amino acid sequence from 1434 to 1459, amino acid sequence from 1585 to 1659, amino acid sequence from 1857 to 2455, amino acid sequence from 2544 to 2561, amino acid sequence from 2603 to 2776, amino acid sequence from 3065 to 3092 , 3124 to 3470, and 3587 to 3689, or a functionally equivalent amino acid sequence thereof. The PH domain derived from the AGAP3 gene includes, for example, the amino acid sequence at positions 404 to 465 of SEQ ID NO: 20, or a functionally equivalent amino acid sequence thereof. The RING domain derived from the TRIM24 gene includes, for example, the amino acid sequence at positions 56 to 82 of SEQ ID NO: 22, or a functionally equivalent amino acid sequence thereof. The kinase domain derived from the BRAF gene includes, for example, the amino acid sequence at positions 457 to 717 of SEQ ID NO: 24, or a functionally equivalent amino acid sequence thereof.

  The fusion gene of ARMC10 gene and BRAF gene may preferably be a polynucleotide comprising the nucleotide sequence of the fusion gene of human ARMC10 gene and human BRAF gene represented by SEQ ID NO: 43, or a functionally equivalent nucleotide sequence thereof. . Further, the polypeptide encoded by the fusion gene of ARMC10 gene and BRAF gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 44 or a functionally equivalent amino acid sequence thereof.

  The fusion gene of AKAP9 gene and BRAF gene may preferably be a polynucleotide comprising the base sequence of the fusion gene of human AKAP9 gene and human BRAF gene represented by SEQ ID NO: 45, or a functionally equivalent base sequence thereof. . In addition, the polypeptide encoded by the fusion gene of AKAP9 gene and BRAF gene may be an amino acid sequence represented by SEQ ID NO: 46, or a polypeptide comprising a functionally equivalent amino acid sequence.

  The fusion gene of AGAP3 gene and BRAF gene may preferably be a polynucleotide comprising the base sequence of the fusion gene of human AGAP3 gene and human BRAF gene represented by SEQ ID NO: 47, or a functionally equivalent base sequence thereof. . Further, the polypeptide encoded by the fusion gene of AGAP3 gene and BRAF gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 48 or a functionally equivalent amino acid sequence thereof.

  The fusion gene of TRIM24 gene and BRAF gene may preferably be a polynucleotide comprising the nucleotide sequence of the fusion gene of human TRIM24 gene and human BRAF gene represented by SEQ ID NO: 49, or a functionally equivalent nucleotide sequence thereof. . Further, the polypeptide encoded by the fusion gene of TRIM24 gene and BRAF gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 50 or a functionally equivalent amino acid sequence thereof.

  For example, the base sequences functionally equivalent to the base sequences represented by SEQ ID NOs: 15, 17, 19, 21, 23, 43, 45, 47 or 49 are SEQ ID NOs: 15, 17, 19, 21, 23, 43, respectively. , 45, 47 or 49, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the nucleotide sequence having the identity, Alternatively, it may be a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence represented by SEQ ID NO: 15, 17, 19, 21, 23, 43, 45, 47, or 49. . In addition, amino acid sequences functionally equivalent to the amino acid sequences represented by SEQ ID NOs: 16, 18, 20, 22, 24, 44, 46, 48, or 50 are SEQ ID NOs: 16, 18, 20, 22, 24, respectively. Amino acids having the identity of, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence represented by 44, 46, 48, or 50 Or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown by SEQ ID NO: 16, 18, 20, 22, 24, 44, 46, 48, or 50 It may be.

  Similarly, the amino acid sequence of positions 55 to 140 of SEQ ID NO: 16, the amino acid sequence of positions 164 to 914 of SEQ ID NO: 18, the amino acid sequence of positions 944 to 1022, the amino acid sequence of positions 1100 to 1185, and the amino acid sequence of positions 1253 to 1280 , Amino acid sequence from 1336 to 1392, amino acid sequence from 1434 to 1459, amino acid sequence from 1585 to 1659, amino acid sequence from 1857 to 2455, amino acid sequence from 2544 to 2561, amino acid sequence from 2603 to 2776, 3065 -3093 amino acid sequence, 3124-3470 amino acid sequence, and 3587-3689 amino acid sequence, SEQ ID NO: 20, 404-465 amino acid sequence, SEQ ID NO: 56, amino acid sequence 56-82, SEQ ID NO: The amino acid sequences functionally equivalent to the amino acid sequences of positions 457 to 717 of 24 are the amino acid sequence of positions 55 to 140 of SEQ ID NO: 16, the amino acid sequence of positions 164 to 914 of SEQ ID NO: 18, and the positions of positions 944 to 1022, respectively. Amino acid sequence, amino acids 1100 to 1185, amino acids 1253 to 1280 Acid sequence, amino acid sequence from 1336 to 1392, amino acid sequence from 1434 to 1459, amino acid sequence from 1585 to 1659, amino acid sequence from 1857 to 2455, amino acid sequence from 2544 to 2561, amino acid sequence from 2603 to 2776 , Amino acid sequence of positions 3065-3092, amino acid sequence of positions 3124-3470, and amino acid sequence of positions 3587-3689, amino acid sequence of positions 404-465 of SEQ ID NO: 20, amino acid sequence of positions 56-82 of SEQ ID NO: 22, An amino acid sequence having identity of, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence at positions 457 to 717 of SEQ ID NO: 24, Or the amino acid sequence of positions 55 to 140 of SEQ ID NO: 16, the amino acid sequence of positions 164 to 914 of SEQ ID NO: 18, the amino acid sequence of positions 944 to 1022, the amino acid sequence of positions 1100 to 1185, the amino acid sequence of positions 1253 to 1280, 1336 ~ Amino acid sequence of 1392, amino acid sequence of 1434 to 1459, amino acid sequence of 1585 to 1659 Amino acid sequence, amino acid sequence from position 1857 to 2455, amino acid sequence from position 2544 to 2561, amino acid sequence from position 2603 to 2776, amino acid sequence from position 3065 to 3092, amino acid sequence from position 3124 to 3470, and position from 3587 to 3689 Amino acid sequence, amino acid sequence of positions 404 to 465 of SEQ ID NO: 20, amino acid sequence of positions 56 to 82 of SEQ ID NO: 22, amino acid sequence of positions 457 to 717 of SEQ ID NO: 24, one or more amino acids are added or deleted And / or a substituted amino acid sequence.

(V) Fusion gene of LMNA gene or TPM3 gene and NTRK1 gene In one embodiment, the marker for colorectal cancer detection of the present invention is a fusion gene of LMNA (lamin A / C) gene and NTRK1 (neurotrophic receptor tyrosine kinase 1) gene Or a fusion gene of TPM3 (tropomyosin 3) gene and NTRK1 gene, or a polypeptide encoded by the fusion gene.

  In the present specification, the LMNA gene may preferably be a polynucleotide comprising the base sequence of the human LMNA gene represented by SEQ ID NO: 25, or a functionally equivalent base sequence thereof. The protein encoded by the LMNA gene may be a polypeptide containing the amino acid sequence of the human LMNA protein represented by SEQ ID NO: 26, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the TPM3 gene may preferably be a polynucleotide comprising the base sequence of the human TPM3 gene represented by SEQ ID NO: 27, or a functionally equivalent base sequence thereof. Further, the protein encoded by the TPM3 gene may be a polypeptide containing the amino acid sequence of the human TPM3 protein represented by SEQ ID NO: 28, or a functionally equivalent amino acid sequence thereof.

  In the present specification, the NTRK1 gene is preferably a base sequence of the human NTRK1 gene represented by SEQ ID NO: 29, for example, 80% or more, preferably 90% or more, 95% or more with respect to the base sequence represented by SEQ ID NO: 29. , 97% or more, 98% or more, or 99% or more of the nucleotide sequence, or a polynucleotide containing a functionally equivalent nucleotide sequence thereof. Further, the protein encoded by the NTRK1 gene may be a polypeptide containing the amino acid sequence of the human NTRK1 protein represented by SEQ ID NO: 30, or a functionally equivalent amino acid sequence thereof.

  The fusion gene is not limited as long as it has canceration ability, but includes the N-terminal sequence of the protein encoded by the LMNA gene or TPM3 gene on the N-terminal side, and the C-terminal sequence of the protein encoded by the NTRK1 gene Those encoding a polypeptide containing at the C-terminal side are preferred.

  The fusion gene preferably encodes a polypeptide comprising a portion of a coiled-coil domain derived from a protein encoded by the LMNA gene or a protein encoded by the TPM3 gene, and a kinase domain derived from the NTRK1 gene.

  The coiled coil domain derived from the LMNA gene includes, for example, at least one of the amino acid sequence at positions 34 to 218 and the amino acid sequence at positions 243 to 383 of SEQ ID NO: 26, or a functionally equivalent amino acid sequence thereof. A part of the coiled coil domain derived from the TPM3 gene includes, for example, the amino acid sequence at positions 1 to 211 of SEQ ID NO: 28, or a functionally equivalent amino acid sequence thereof. In addition, the kinase domain derived from the NTRK1 gene includes, for example, the amino acid sequence at positions 510 to 781 of SEQ ID NO: 30, or a functionally equivalent amino acid sequence thereof.

  The fusion gene of LMNA gene and NTRK1 gene may preferably be a polynucleotide comprising the base sequence of the fusion gene of human LMNA gene and human NTRK1 gene represented by SEQ ID NO: 51, or a functionally equivalent base sequence thereof. . The protein encoded by the fusion gene of LMNA gene and NTRK1 gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 52 or a functionally equivalent amino acid sequence thereof.

  The fusion gene of TPM3 gene and NTRK1 gene may preferably be a polynucleotide comprising the base sequence of the fusion gene of human TPM3 gene and human NTRK1 gene represented by SEQ ID NO: 53, or a functionally equivalent base sequence thereof. . Further, the protein encoded by the fusion gene of TPM3 gene and NTRK1 gene may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 54 or a functionally equivalent amino acid sequence thereof.

  For example, the base sequence functionally equivalent to the base sequence represented by SEQ ID NO: 25, 27, 29, 51 or 53 is, for example, the base sequence represented by SEQ ID NO: 25, 27, 29, 51 or 53, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or a nucleotide sequence having an identity of 99% or more, or a base represented by SEQ ID NO: 25, 27, 29, 51 or 53 It may be a base sequence in which one or more bases are added, deleted, and / or substituted in the sequence. The amino acid sequence functionally equivalent to the amino acid sequence represented by SEQ ID NO: 26, 28, 30, 52, or 54 is the amino acid sequence represented by SEQ ID NO: 26, 28, 30, 52, or 54, respectively. An amino acid sequence having, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more, or SEQ ID NO: 26, 28, 30, 52, or 54 It may be an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the amino acid sequence shown.

  Similarly, the amino acid sequence of positions 34 to 218 of SEQ ID NO: 26, the amino acid sequence of positions 243 to 383 of SEQ ID NO: 26, the amino acid sequence of positions 1 to 211 of SEQ ID NO: 28, or the amino acids of positions 510 to 781 of SEQ ID NO: 30 The amino acid sequence functionally equivalent to the sequence is the amino acid sequence of positions 34 to 218 of SEQ ID NO: 26, the amino acid sequence of positions 243 to 383 of SEQ ID NO: 26, the amino acid sequence of positions 1 to 211 of SEQ ID NO: 28, or An amino acid sequence having identity of, for example, 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence at positions 510 to 781 of SEQ ID NO: 30, Alternatively, in the amino acid sequence of positions 34 to 218 of SEQ ID NO: 26, the amino acid sequence of positions 243 to 383 of SEQ ID NO: 26, the amino acid sequence of positions 1 to 211 of SEQ ID NO: 28, or the amino acid sequence of positions 510 to 781 of SEQ ID NO: 30 Amino acids with one or more amino acids added, deleted, and / or substituted It may be an array.

  By detecting the colorectal cancer detection marker of the present invention, the presence or absence of colorectal cancer injury in a sample of colorectal cancer, for example, microsatellite unstable colorectal cancer (MSI-H CRC), is determined. be able to. In MSI-H CRC, since the chromosome was relatively stable and the abundance ratio of the fusion gene was considered to be not so high, the polynucleotide constituting the fusion gene or the polypeptide encoded by the polynucleotide was designated as MSI. It was surprising that it could be used to detect -H CRC.

  The marker for colorectal cancer detection of the present invention may be used alone or in combination. Further, the colorectal cancer detection marker of the present invention is used in combination with other existing colorectal cancer detection markers such as CEA gene, CA19-9 gene, or NCC-ST-439 gene or proteins encoded by these. Also good. Furthermore, the colorectal cancer detection marker of the present invention may be used in combination with other colorectal cancer detection methods such as fecal occult blood test. Colorectal cancer detection accuracy can be increased by combination with other markers or with other methods.

<Means and kits for detecting markers>
In one aspect, the present invention relates to a kit for detecting a marker for detecting colorectal cancer described herein, such as a primer set, a probe, an aptamer, an antibody, or at least one of these. These can be easily designed by those skilled in the art according to the marker.

1. Primer set The primer set is not particularly limited as long as it can specifically detect each of the above-mentioned markers for detecting colorectal cancer, in particular, the protein or gene mutation part of each marker. For example, the markers include the SLC12A2 gene and the INSR gene. If it is a fusion gene of
The forward primer is composed of nucleotides comprising 14 to 30 consecutive nucleotides of SEQ ID NO: 33, such as 16 to 28 nucleotides, preferably 18 to 26 nucleotides, and the reverse primer is 14 to 14 consecutive nucleotides complementary to the sequence of SEQ ID NO: 33. 14 to 30 bases consisting of nucleotides comprising 30 bases, for example, 16 to 28 bases, preferably 18 to 26 bases, or the forward primer hybridizes under stringent conditions to a nucleic acid consisting of a complementary sequence of SEQ ID NO: 33 Consisting of nucleotides comprising, for example, 16 to 28 bases, preferably 18 to 26 bases, and the reverse primer hybridizes under stringent conditions to the nucleic acid comprising the sequence of SEQ ID NO: 33, for example, 16 to 28 Consisting of nucleotides, preferably nucleotides comprising a sequence of 18-26 bases,
Includes forward and reverse primers.

  In the present specification, the “stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For stringent conditions, known hybridization conditions can be used. For example, it may be determined appropriately with reference to Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions may be set according to the hybridization method temperature and the salt concentration contained in the solution, and the temperature and the salt concentration contained in the solution in the washing step of the hybridization method. More detailed stringent conditions include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 × SSC (83 mM NaCl, 83 mM sodium citrate) and a temperature of 42 ° C. may be mentioned.

  When the marker is a RUFY1 gene or a fusion gene of NCOA4 gene and RET gene, a primer set can be designed in the same manner based on the nucleotide sequence shown in SEQ ID NO: 35 or 37, respectively. When the marker is a KANK1 gene or a fusion gene of EML4 gene and NTRK3 gene, a primer set can be designed in the same manner based on the nucleotide sequence represented by SEQ ID NO: 39 or 41, respectively. When the marker is ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, the primer set is similarly set based on the nucleotide sequence shown in SEQ ID NO: 43, 45, 47, or 49, respectively. Can be designed. When the marker is an LMNA gene or a fusion gene of TPM3 gene and NTRK1 gene, a primer set can be designed in the same manner based on the nucleotide sequence represented by SEQ ID NO: 51 or 53, respectively.

2. Probe The probe for detecting the marker is not particularly limited as long as it can detect the marker, particularly a protein or gene mutation part of the marker. For example, when the marker is a fusion gene of SLC12A2 gene and INSR gene, the probe is
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 33, at least 14, for example 20, preferably 30, or a complementary sequence thereof,
It may be a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising at least 14, for example, 20, preferably 30 nucleotide sequences of SEQ ID NO: 33 or a complementary sequence thereof.

  When the marker is a RUFY1 gene or a fusion gene of NCOA4 gene and RET gene, a probe can be designed in the same manner based on the nucleotide sequence represented by SEQ ID NO: 35 or 37, respectively. When the marker is a KANK1 gene or a fusion gene of the EML4 gene and the NTRK3 gene, a probe can be similarly designed based on the nucleotide sequence represented by SEQ ID NO: 39 or 41, respectively. When the marker is ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, the probe is similarly designed based on the nucleotide sequence shown in SEQ ID NO: 43, 45, 47, or 49, respectively. can do. When the marker is an LMNA gene or a fusion gene of TPM3 gene and NTRK1 gene, a probe can be similarly designed based on the nucleotide sequence shown in SEQ ID NO: 51 or 53, respectively.

  Primers and probes can be prepared by known methods known to those skilled in the art, and are not limited, and can be prepared by, for example, chemical synthesis methods.

3. Aptamer The aptamer for the marker can be prepared by a known method such as SELEX (systematic evolution of ligands by exponential enrichment). In the SELEX method, a nucleic acid molecule bound to a target molecule is selected from a pool consisting of a random sequence region and nucleic acid molecules having primer binding regions at both ends, amplified after recovery, and then transferred to the nucleic acid pool of the next round. This is a method of selecting a nucleic acid having a stronger binding force to a target molecule by repeating a series of cycles of several to several tens of rounds (for example, Tuerk C, Gold L (1990) Science 249 (4968): 505- See 510).

4). Antibody The antibody that specifically binds to the marker may be a polyclonal antibody, a monoclonal antibody, a recombinant antibody, or a synthetic antibody. “Polyclonal antibody” refers to a group of a plurality of immunoglobulins that recognize and bind different epitopes of the same antigen. Polyclonal antibodies can be obtained from the animal's serum after immunization of the animal with the target molecule as an antigen. “Monoclonal antibody” refers to a group of clones of a single immunoglobulin. A monoclonal antibody can be produced, for example, by using a hybridoma obtained by isolating a B cell producing an antibody from an immunized animal and fusing it with a myeloma cancer cell. As used herein, “recombinant antibody” means an antibody produced by combining amino acid sequences of antibodies from different animals such as chimeric antibodies and humanized antibodies. In the present specification, “synthetic antibody” means an antibody synthesized by a chemical method or a recombinant DNA method, and includes a single chain fragment of variable region (scFv), a diabody, Examples include triabody and tetrabody.

  As the primer set, probe, aptamer, or antibody, one that detects the fusion part of the fusion gene or fusion polypeptide constituting the marker described in the present specification may be used alone. Alternatively, the markers described in the present specification can be detected using a combination of a plurality of those that detect a non-fusion portion.

  In one embodiment, the invention relates to a kit comprising means for detecting a marker for colorectal cancer detection described herein. The kit of the present invention may contain at least one of, for example, a buffer, an enzyme, and an instruction manual in addition to the primer set, probe, aptamer, or antibody.

<Determination method of colorectal cancer>
In one embodiment, the present invention relates to a method for assisting in the determination of the presence or absence of colorectal cancer or the possibility of colorectal cancer. The colorectal cancer to be diagnosed in this embodiment may be any of cecal cancer, colon cancer, and rectal cancer.

  The method includes a step (detection step) of detecting a marker for colorectal cancer detection described in the present specification in a sample obtained from a subject. The step of detecting a marker for detecting colorectal cancer is performed by a known gene analysis method (for example, PCR, RT-PCR, Real-time PCR, LCR (Ligase chain reaction), LAMP (Loop-Loop- mediated isothermal amplification) method, microarray method, Northern hybridization method, dot blot method, fluorescent in situ hybridization (FISH) method, analysis by next-generation sequencer, etc.). For example, using a nucleic acid extracted from a sample derived from a subject, for example, mRNA, a gene amplification technique using an appropriate primer, a hybridization technique using an appropriate probe, or the like is used. Although a detection process is not limited, you may detect using the said means for detecting a marker, for example, a kit containing a primer set, a probe, an aptamer, an antibody, or any of these, for example.

  In addition to the detection step, the method determines that the subject is suffering from colon cancer or has a high possibility of suffering from colon cancer when the colorectal cancer detection marker described herein is detected. The process (determination process) to include is included as an arbitrary process.

  In the present specification, the “sample” means a biological sample subjected to the method of the present invention. Examples of samples that can be used in the present invention include, but are not limited to, cells or tissues isolated from living organisms. Examples of tissues include colon cancer lesions, such as the cecum, colon, and rectum, for example, biopsy samples of these tissues can be used.

  The method for determining the incidence of colorectal cancer according to the present invention can be used in combination with other methods (for example, X-ray photography, endoscopy, etc.). The accuracy of the discrimination method of the present invention can be further enhanced by combining with other disease discrimination methods or diagnosis methods.

<Pharmaceutical composition and treatment and / or prevention method>
In one aspect, the present invention relates to a pharmaceutical composition for treating and / or preventing colorectal cancer in a subject. The pharmaceutical composition of the present invention contains a fusion gene expression inhibitor or kinase inhibitor described herein as an active ingredient.

  In one aspect, the present invention treats and / or treats colorectal cancer in a subject, comprising administering to the subject an inhibitor or kinase inhibitor of a fusion gene described herein, or the pharmaceutical composition described above. It relates to a method for prevention.

  The pharmaceutical composition or colorectal cancer treatment and / or prevention method of the present invention comprises (i) a fusion gene of any gene and INSR gene, (ii) a fusion gene of RUFY1 gene or NCOA4 gene and RET gene, (iii) KANK1 A gene or a fusion gene of EML4 gene and NTRK3 gene, (iv) a fusion gene of ARMC10 gene, AKAP9 gene, AGAP3 gene, or TRIM24 gene and BRAF gene, and (v) a fusion gene of LMNA gene or TPM3 gene and NTRK1 gene Applies to subjects having at least one fusion gene selected from the group consisting of: Since each gene and each fusion gene described in (i) to (v) are as described in the above <Colorectal cancer detection marker>, description is omitted.

  The expression inhibitor or kinase inhibitor of the fusion gene that can be contained in the pharmaceutical composition of the present invention and that can be used in the method for treating and / or preventing colorectal cancer of the present invention is described below.

  The fusion gene expression inhibitor described herein may be an RNAi molecule. As used herein, “RNAi molecule” refers to inducing RNAi (RNA interference) in vivo and suppressing (silencing) the expression of the gene through degradation of the target gene transcript. An RNA molecule that can be produced (Fire A. et al., 1998, Nature, 391, 806-811). Examples of RNAi molecules include siRNA or shRNA. In this specification, “siRNA” (small interference RNA) means a sense strand (passenger strand) having a base sequence corresponding to a part of a target gene, and an antisense strand (guide strand) thereof. It is a small double-stranded RNA. In this specification, “shRNA” (short hairpin RNA) means a single-stranded RNA in which the sense strand and the antisense strand of the above siRNA or mature double-stranded miRNA are linked by a short spacer sequence having an appropriate sequence. Say. In other words, shRNA has a hairpin stem-loop as a whole molecule by forming a stem structure by pairing the sense region and the antisense region with each other in one molecule and simultaneously forming a loop structure with the spacer sequence. Forming a structure. The siRNA or shRNA for the fusion gene can be appropriately designed by those skilled in the art based on the sequence of the fusion gene. The RNAi molecule is preferably introduced into a vector and administered to the subject.

  When the subject has the fusion gene described in (i), the kinase inhibitor is an inhibitor of a kinase derived from a protein encoded by the INSR gene (also referred to as “INSR inhibitor”), and (ii) When having the described fusion gene, the kinase inhibitor is an inhibitor of a kinase derived from the protein encoded by the RET gene (also referred to as “RET inhibitor”), and having the fusion gene according to (iii) The kinase inhibitor is an inhibitor of a kinase derived from a protein encoded by the NTRK3 gene (also referred to as “NTRK3 inhibitor”). When the kinase inhibitor has the fusion gene described in (iv), the kinase inhibitor is a BRAF gene. An inhibitor of a kinase derived from the protein encoded by (also referred to as “BRAF inhibitor”), and having the fusion gene described in (v), Over peptidase inhibitor is an inhibitor of the kinase derived from a protein encoded by the NTRK1 gene (also referred to as "NTRK1 inhibitors"). The kinase inhibitor may be at least one drug selected from the group consisting of aptamers, neutralizing antibodies, and low molecular weight compounds. About the preparation method of an aptamer and (neutralization) antibody, it applies to the method described in the above <means for detecting a marker>. Hereinafter, low molecular weight compounds that can be used as kinase inhibitors are exemplified.

  Examples of INSR inhibitors include linsitinib, NT157, GSK1838705A, NVP-AEW541, BMS-754807, GSK1904529A, NVP-ADW742, BMS-536924, BI 885578, and Tyrphostin (tylophostin).

  Examples of RET inhibitors include regorafenib (regorafenib), vandetanib (vandetanib), Lenvatinib (renbatinib), Danusertib (danuserib), Alectinib (Alectinib), TG101209, SML1435, TG101348 (Fedratinib), 3- (Fedratinibdi) Ridenyl) indoline-2-one), Carbozantinib (carbozantinib), and ML 786 dihydrochloride.

  Examples of NTRK3 inhibitors include entrectinib (entrectinib), GNF-5837, MK-5108 (VX-689), Tepotinib (tepotinib), LOXO-101, MK-2461, Danusertib, BMS-777607, BMS-754807 , PF-03814735, SNS-314 Mesylate, AG-1478 (Tyrphostin AG-1478), CEP-751, Lestaurtinib (Restaurtinib) (CEP-701), DS-6051b, crizotinib (crizotinib), PLX7486, Dovitinib (dobitinib) ( TKI-258, CHIR-258).

  Examples of BRAF inhibitors include trametinib, selumetinib, selumetinib, PD0325901, U0126-EtOH, PD184352 (CI-1040), PD98059, BIX 02189, Pimasertib (Bimaceltib), BIX 02188, TAK-733, AZD8330, Binimetinib (Vinimethinib), PD318088, Honokiol (Honokiol), SL-327, RDEA119, GDC-0623, Cobimetinib (Cobimetinib), BI-847325, refametinib (Lefametinib), PLX8394, PLX7904, vemurafenib (Bemurafenib), fen ib, Bra ), RO5126766, CEP-32496, sorafenib (sorafenib), PLX-4720, MLN2480, GDC-0879, RAF265 (CHIR-265), GW5074, SB590885, TAK-632, Encorafenib (LGX818), and LY3009120 It is done.

  Examples of NTRK1 inhibitors include entrectinib, GNF-5837, MK-5108 (VX-689, Tepotini, LOXO-101, MK-246, Danusertib, BMS-77760, BMS-75480, PF-0381473, SNS- 314 mesylate, GW441756, AG-1478 (Tyrphostin AG-1478), CEP-751, Lestaurtinib (CEP-701), DS-6051b, crizotinib, PLX7486, and Dovitinib (TKI-258, CHIR-258).

  The pharmaceutical composition of the present invention may contain two or more of the above active ingredients, or may contain other active ingredients.

  In principle, the pharmaceutical composition of the present invention can be formulated by a method known in the art. For example, it can be formulated using the method described in Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.). The specific formulation method varies depending on the administration method. The administration method is roughly classified into oral administration and parenteral administration, and the administration method can be appropriately selected.

  When the pharmaceutical composition of the present invention is orally administered, a pharmaceutically acceptable carrier may be added. “Pharmaceutically acceptable carrier” refers to a substance that is added in a range that facilitates formulation of a drug and application to a living body and does not inhibit or suppress the action of its active ingredients. For example, an excipient, a binder, a disintegrant, a filler, an emulsifier, a fluid addition modifier, or a lubricant can be used.

  Examples of the “excipient” include sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides (specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, Including dextrin, maltodextrin, starch and cellulose), metal salts (eg, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate), citric acid, tartaric acid, glycine, low, medium, high molecular weight polyethylene glycol (PEG), Pluronic or a combination thereof.

  Examples of the “binder” include starch paste using corn, wheat, rice, or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose sodium and / or polyvinylpyrrolidone.

  Examples of the “disintegrant” include the starch, carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, alginic acid, sodium alginate, or salts thereof.

  Examples of the “filler” include the sugar and / or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).

  Examples of the “emulsifier” include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.

  Examples of the “flow addition modifier” and “lubricant” include silicate, talc, stearate or polyethylene glycol.

  Examples of the dosage form of the oral preparation include solid preparations (including tablets, pills, sublingual tablets, capsules, and drop preparations), granules, powders, powders, and liquids. Furthermore, the solid preparation can be made into a dosage form known in the art, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary. . The specific shape and size of the dosage form are not particularly limited as long as each dosage form is within the range of dosage forms known in the art.

  When the pharmaceutical composition of the present invention is administered parenterally, specific examples thereof include administration by injection. When the pharmaceutical composition of the present invention is administered by injection, it can be prepared as a suspension mixed with a pharmaceutically acceptable solvent and, if necessary, added with a pharmaceutically acceptable carrier.

  The “pharmaceutically acceptable solvent” may be either water or other pharmaceutically acceptable aqueous solution, or an oily liquid. Examples of the aqueous solution include isotonic solutions containing physiological saline, glucose and other adjuvants. Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and low concentrations of nonionic surfactants (eg, polysorbate 80 (TM), HCO-60), polyoxyethylene Examples include sorbitan fatty acid esters. Examples of the oily liquid include sesame oil and soybean oil. As a solubilizing agent, for example, benzyl benzoate or benzyl alcohol can be used in combination. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant.

  Injectables are combined in a pharmaceutically acceptable excipient, emulsifier, suspending agent, surfactant, stabilizer, pH adjuster, etc., in appropriate combinations in unit dosage forms generally required for pharmaceutical practice. It is sufficient to formulate by doing so.

  Injection includes, for example, intravascular injection, intralymphatic injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., and intravascular administration such as intravascular injection or intralymphatic injection, which is systemic administration, is preferable. It may be a local administration that is administered directly.

  The content of the active ingredient in the pharmaceutical composition of the present invention is basically such that the active ingredient can reach the target site in a single administration, and has little or no adverse side effects on the subject to which it is applied. Any amount is sufficient. Such content varies depending on the degree of disease progression, the type of active ingredient, the dosage form and administration method of the pharmaceutical composition, etc., but is appropriately determined by those skilled in the art.

<Fusion gene or polypeptide>
In one embodiment, the present invention provides a fusion gene of SLC12A2 gene and INSR gene, a fusion gene of RUFY1 gene and RET gene, a fusion gene of AKAP9 gene and BRAF gene, or a polypeptide having canceration ability encoded by the fusion gene About.

  The fusion gene of the present invention is, for example, 80% or more, preferably 90% or more with respect to the base sequence represented by SEQ ID NO: 33, 35, or 45, or the base sequence represented by SEQ ID NO: 33, 35, or 45, 95% or more, 97% or more, 98% or more, or 99% or more of the nucleotide sequence, or one or more bases added or deleted in the nucleotide sequence represented by SEQ ID NO: 33, 35, or 45 And / or a substituted base sequence.

  Further, the polypeptide encoded by the fusion gene of the present invention is an amino acid sequence represented by any one of SEQ ID NOs: 34, 36, and 46, and an amino acid sequence represented by any one of SEQ ID NOs: 34, 36, and 46. For example, an amino acid sequence having 80% or more, preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity, or any one of SEQ ID NOs: 34, 36, and 46 It encodes a polypeptide comprising an amino acid sequence in which one or several amino acids have been added, deleted, and / or substituted in the amino acid sequence shown.

<Materials and methods>
material
Tumors resected from CRC (colon cancer) patients were analyzed with informed consent. As a control, normal intestinal mucosa near the tumor was used. This experiment was approved by the Ethics Committee of the University of Tokyo and Teikyo University.

Transcriptome Total RNA was extracted with RNA Bee Reagent (Tel-Test Inc., Friendswood, TX) using RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with DNaseI (Qiagen). RNA integrity was examined using Bioanalyzer (Agilent Technologies) or TapeStation (Agilent Technologies). RNA with a high RNA integrity number was prepared from RNA-seq using a NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, Mass.) Prepared from a polyA-selected RNA. For RNA having a low RNA integrity number, complementary DNA (cDNA) was prepared using random primers and subjected to RNA-seq using TruSeq RNA Access Library Prep Kit (illumina). The prepared RNA-seq library was subjected to 120 bp NGS sequencing from both ends. The fusion gene was detected by deFuse pipeline (https://bitbucket.org/dranew/defuse) and STAR (https://github.com/alexdobin/STAR).

Cloning of the fusion gene The wild type protein and the cDNA of the fusion protein were amplified by reverse transcription polymerase chain reaction (RT-PCR) from the RNA sample and ligated into the pMXs retroviral vector (Cell Biolabs). The sequence of all cDNAs was confirmed by Sanger sequencing.

3T3 Transformation Assay Human embryonic kidney (HEK) 293T cells and mouse 3T3 fibroblasts were obtained from the American Type Culture Collection and Dulbecco's Modified Eagle Medium (DMEM) -F12 supplemented with 10% fetal bovine serum (FBS) Also maintained by Invitrogen). The pMXs vector was introduced into HEK293T cells by transfection together with ecotropic packaging plasmid (Takara Bio) to obtain infectious virus particles. In the focus formation assay, 3T3 cells were infected with ecotropic recombinant retrovirus and cultured in Dulbecco's Modified Eagle Medium-F12 (both Invitrogen) supplemented with 5% calf serum.

Kinase inhibitors Kinase inhibitors (Lintinib, Regorafenib, Entrectinib, and PLX7904) were obtained from Selleck Chemicals.

Statistical analysis Numerical values are shown by median and range. Comparison of numerical values between groups was performed using a nonparametric approach (Wilcoxon rank-sum test). Comparison of the distribution of categorical variables in different groups was performed by Fisher's exact test.

<Result>
Carcinogenic mutations in MSI-H CRC 2836 surgically resected intestinal cancer or adenoma specimens were obtained from patients treated between 1998-2016 at the University of Tokyo Tumor Surgery and Teikyo University Surgery. Out of 2836 tumors, a total of 162 tumors (161 adenocarcinoma and 1 adenoma) are MSI-H (microsatellite instability-high), which shows MSI in both BAT25 and BAT426 in this example. Defined as). In this example, this strict criteria was applied to clearly distinguish MSI-H CRC from microsatellite low instability CRC.

  In the MSI-H CRC (microsatellite unstable colorectal cancer), which was subjected to RNA-seq analysis to identify transcriptionally active mutant alleles, the abundance of fusion genes is so high because the chromosome is relatively stable. I thought it was not. However, contrary to expectation, transcripts encoding fusion kinases were found in 111 tumors (33 LS / s) by RNA-seq analysis with poly-A capture RNA-seq (n = 94) or random primers (n = 17). LL tumors, 72 MM tumors, and 6 subtype unknown tumors) were found in 11 MM tumors. Interestingly, the fusion kinase was mutually exclusive with the KRAS and BRAF mutations. A schematic diagram of the protein encoded by the detected fusion gene is shown in FIG. 1, and the sequence information of the detected fusion gene is shown in Table 1 below.

  For all fusion genes including the novel fusion gene SLC12A2-INSR, the expression of the fusion transcript was confirmed by reverse transcription polymerase chain reaction (RT-PCR) (data not shown). For 8 fusion genes including SLC12A2-INSR, the fusion points on the genome were identified by long range PCR (data not shown).

  Subsequently, when the fusion gene was introduced into the cells and 3T3 transformation assay was performed, it was confirmed that each fusion gene shown in the figure had tumorigenicity (FIG. 2).

  Furthermore, inhibitors that inhibit the activity of each kinase contained in the proteins encoded by these fusion genes reduced malignant transformation of 3T3 cells expressing the fusion protein in a concentration-dependent manner (FIG. 3). For comparison, a similar test was performed for v-ras, which is a virus-derived oncogene. These data suggest that kinase inhibitors can be therapeutic agents for tumors caused by fusion proteins.

Oncogenic fusion genes in MSS CRC
To examine whether MSS CRC (microsatellite stable colorectal caner) also has a carcinogenic fusion gene, we used RNA-seq data from the MSS dataset of The Cancer Genome Atlas (TCGA). analyzed. 26 paired-end RNA-seq data and 181 single-read RNA-seq data (median 36213120 reads per sample, 76 bp long) were available. As a result of the analysis, no oncogenic fusion gene related to tyrosine kinase was detected in the RNA-seq data from the MSS CRC data set. On the other hand, the same conditions for MSI colorectal cancer (36 mega reads per sample randomly selected from the above-mentioned MSI-H CRC RNA-seq data were reduced to 76 bp length to adjust the analysis conditions, and used for analysis) As a result, 10 out of 11 fusion genes were found. These data suggest that oncogenic fusion genes for tyrosine kinases were more frequent than MSS CRC in MSI-CRC, especially in the MM subtype.

  According to the present invention, a marker capable of detecting colon cancer rapidly and accurately is provided. The present invention also provides a pharmaceutical composition for effectively treating and / or preventing colorectal cancer. Since the number of patients with colorectal cancer and the number of deaths due to colorectal cancer are very large, the present invention has great industrial significance.

Claims (15)

The following (i) to (v):
(I) a fusion gene of any gene and INSR gene,
(Ii) RUFY1 gene or NCOA4 gene and RET gene fusion gene,
(Iii) KANK1 gene or EML4 gene and NTRK3 gene fusion gene,
(Iv) ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, and (v) a fusion gene of LMNA gene or TPM3 gene and NTRK1 gene,
A marker for detecting colorectal cancer comprising a fusion gene selected from the group consisting of: or a polypeptide encoded by the fusion gene.   The INSR gene, RUFY1 gene, NCOA4 gene, RET gene, KANK1 gene, EML4 gene, NTRK3 gene, AMRC10 gene, AKAP9 gene, AGAP3 gene, TRIM24 gene, BRAF gene, LMNA gene, TPM3 gene, and NTRK1 gene are respectively arranged. The polypeptide according to claim 1, which encodes a polypeptide comprising the amino acid sequence shown by the numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. marker.   The marker according to claim 1 or 2, wherein the arbitrary gene described in (i) is the SLC12A2 gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32. The fusion gene according to (i) encodes a polypeptide containing a kinase domain derived from the INSR gene,
The fusion gene described in (ii) encodes a polypeptide comprising a coiled coil domain of the RUFY1 gene or a part thereof or a coiled coil domain derived from the NCOA4 gene, and a kinase domain derived from the RET gene,
The fusion gene according to (iii) encodes a polypeptide comprising a coiled coil domain derived from the KANK1 gene or EML4 gene, and a kinase domain derived from the NTRK3 gene,
(Iv) the fusion gene described in (a) ARMC10 gene-derived ARM domain, AKAP9 gene-derived coiled-coil domain, AGAP3 gene-derived PH domain, or TRIM24 gene-derived RING domain, and (b) BRAF gene-derived Encodes a polypeptide comprising a kinase domain,
(V) The marker according to any one of claims 1 to 3, which encodes a polypeptide comprising a part of a coiled-coil domain derived from LMNA gene or TPM3 gene and a kinase domain derived from NTRK1 gene. In (i), the kinase domain derived from the INSR gene contains the amino acid sequence of positions 1011 to 1286 of SEQ ID NO: 2,
In (ii), the coiled coil domain derived from the RUFY1 gene or a part thereof includes the amino acid sequences of positions 321 to 374 and / or 405 to 553 of SEQ ID NO: 4, and the coiled coil domain derived from the NCOA4 gene is represented by 1 to Comprising the amino acid sequence at position 200, the kinase domain derived from the RET gene comprising the amino acid sequence at positions 724 to 1016 of SEQ ID NO: 8,
In (iii), the coiled coil domain derived from the KANK1 gene contains at least one of the amino acid sequences of positions 258 to 316, 361 to 395, and 446 to 500 of SEQ ID NO: 10, and the coiled coil domain derived from the EML4 gene is SEQ ID NO: 12 comprising the amino acid sequence of positions 17 to 51, the kinase domain derived from the NTRK3 gene comprises the amino acid sequence of positions 538 to 825 of SEQ ID NO: 14,
In (iv), the ARM domain derived from the ARMC10 gene contains the amino acid sequence of positions 55 to 140 of SEQ ID NO: 16, the coiled coil domain derived from the AKAP9 gene is the amino acid sequence of positions 164 to 914 of SEQ ID NO: 18, and positions 944 to 1022 Amino acid sequence, amino acid sequence from 1100 to 1185, amino acid sequence from 1253 to 1280, amino acid sequence from 1336 to 1392, amino acid sequence from 1434 to 1459, amino acid sequence from 1585 to 1659, amino acid sequence from 1857 to 2455 AGAP3 gene comprising at least one of amino acid sequences from 2544 to 2561, amino acid sequences from 2603 to 2776, amino acids from 3065 to 3092, amino acids from 3124 to 3470, and amino acids from 3587 to 3689 The derived PH domain includes the amino acid sequence of positions 404 to 465 of SEQ ID NO: 20, the RING domain derived from the TRIM24 gene includes the amino acid sequence of positions 56 to 82 of SEQ ID NO: 22, and the kinase domain derived from the BRAF gene includes SEQ ID NO: 24 Of 457-717 position amino acid sequence,
In (v), the LMNA gene-derived coiled-coil domain includes amino acid sequences of positions 34 to 218 and / or 243 to 383 of SEQ ID NO: 26, and a part of the coiled-coil domain derived from the TPM3 gene is 1-211 of SEQ ID NO: 28 The kinase domain derived from the NTRK1 gene comprises the amino acid sequence of positions 510 to 781 of SEQ ID NO: 30,
The marker according to claim 4. A fusion gene of any gene and the INSR gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 34;
RUFY1 gene or NCOA4 gene and RET gene fusion gene each encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 36 or 38,
A fusion gene of KANK1 gene or EML4 gene and NTRK3 gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 40 or 42, respectively;
ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene each encodes a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 44, 46, 48, or 50,
The marker according to claim 5, wherein the LMNA gene or the fusion gene of TPM3 gene and NTRK1 gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 52 or 54, respectively.   The marker according to any one of claims 1 to 6, wherein the colorectal cancer is microsatellite unstable colorectal cancer.   A kit comprising at least one of a primer set, a probe, an aptamer, or an antibody for detecting the marker according to any one of claims 1 to 7.   A method for assisting in determining the presence or absence of colorectal cancer or the possibility of colorectal cancer, comprising a step of detecting the marker according to any one of claims 1 to 7 in a sample obtained from a subject. The following (i) to (v):
(I) a fusion gene of any gene and INSR gene,
(Ii) RUFY1 gene or NCOA4 gene and RET gene fusion gene,
(Iii) KANK1 gene or EML4 gene and NTRK3 gene fusion gene,
(Iv) ARMC10 gene, AKAP9 gene, AGAP3 gene, or a fusion gene of TRIM24 gene and BRAF gene, and (v) a fusion gene of LMNA gene or TPM3 gene and NTRK1 gene,
A pharmaceutical composition comprising a fusion gene expression inhibitor or a kinase inhibitor for treating colorectal cancer in a subject having at least one fusion gene selected from the group consisting of:
When the subject has the fusion gene described in (i), the kinase inhibitor is an INSR inhibitor;
If the subject has at least one of the fusion genes described in (ii), the kinase inhibitor is a RET inhibitor;
When the subject has at least one of the fusion genes described in (iii), the kinase inhibitor is an NTRK3 inhibitor;
If the subject has at least one of the fusion genes described in (iv), the kinase inhibitor is a BRAF inhibitor;
If the subject has at least one of the fusion genes described in (v), the kinase inhibitor is an NTRK1 inhibitor;
Said pharmaceutical composition.   The INSR gene, RUFY1 gene, NCOA4 gene, RET gene, KANK1 gene, EML4 gene, NTRK3 gene, AMRC10 gene, AKAP9 gene, AGAP3 gene, TRIM24 gene, BRAF gene, LMNA gene, TPM3 gene, and NTRK1 gene are respectively arranged. 11. The polypeptide according to claim 10, which encodes a polypeptide comprising the amino acid sequence indicated by the numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. Pharmaceutical composition.   The pharmaceutical composition according to claim 10 or 11, wherein the arbitrary gene described in (i) is the SLC12A2 gene, and the SLC12A2 gene encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32.   The fusion gene expression inhibitor is an RNAi molecule, or the kinase inhibitor is at least one drug selected from the group consisting of aptamers, neutralizing antibodies, and low molecular weight compounds. Pharmaceutical composition as described in any one of these. The amino acid sequence represented by any of SEQ ID NOs: 34, 36, and 46;
An amino acid sequence having 90% or more identity to the amino acid sequence represented by any of SEQ ID NOs: 34, 36, and 46, or 1 or 2 in the amino acid sequence represented by any of SEQ ID NOs: 34, 36, and 46 A polypeptide comprising an amino acid sequence in which several amino acids have been added, deleted and / or substituted.   A polynucleotide encoding the polypeptide of claim 14.