WO2016152352A1

WO2016152352A1 - Melanoma-specific biomarker and use thereof

Info

Publication number: WO2016152352A1
Application number: PCT/JP2016/054968
Authority: WO
Inventors: 昌志加藤; 伊知朗矢嶋; 湖州恵武田; 友二後藤
Original assignee: 国立大学法人名古屋大学; 学校法人中部大学
Priority date: 2015-03-20
Filing date: 2016-02-20
Publication date: 2016-09-29
Also published as: JPWO2016152352A1

Abstract

The present invention addresses the problem of providing: a marker molecule which enables the highly efficient diagnosis of melanoma; and a novel therapy target molecule which can be utilized in an effective therapy method. As melanoma-specific markers, PDZRN3, DTX3L, CSS3 and BRP44L are provided. Also provided are a test method, a therapy method and others each using these biological molecules.

Description

Melanoma-specific biomarkers and uses thereof

The present invention relates to a melanoma-specific biomarker. Specifically, the present invention relates to a novel biomarker specific to melanoma and a test method using the same. This application claims priority based on Japanese Patent Application No. 2015-058692 filed on Mar. 20, 2015, the entire contents of which are incorporated by reference.

Conventionally, S100, melanomainhibiting activity (MIA), 5-S-cysteinyldopa (5-S-CD), 6-hydroxy-5-methoxyindole-2-carboxylic acid (6-H-5) are used as tumor markers for the diagnosis of melanoma. -MI-2CA) is used (Non-Patent Document 1). On the other hand, Dacarbazine has been used for a long time as a molecular targeted therapeutic drug for melanoma, but recently, new molecular targeted drugs targeting MAP kinase pathway related molecules (BRAF, MEK) have been developed and put into clinical practice. It has appeared (Non-Patent Document 2).

Melanoma has the highest rate of increase among all carcinomas (world statistics). Moreover, melanoma with advanced stage has a poor prognosis, and early detection is important. On the other hand, conventional biomarkers for melanoma are effective only for advanced stage melanoma and are less useful for early diagnosis. If a gene or protein that is highly related to malignant melanoma can be used as a new diagnostic marker, a more reliable diagnosis can be made earlier. Moreover, pathological diagnosis of melanoma is not easy, and if a biomarker highly specific to melanoma can be provided, it can contribute to improvement of pathological diagnosis accuracy.

The effect of dacarbazine, which is frequently used in the treatment of melanoma, is limited, and molecular targeted drugs targeting BRAF are temporarily very effective, but drug resistant melanoma recurs after several months. In addition, these molecular target drugs target specific pathways of intracellular signals, and cannot be applied when the function of melanoma is expressed without going through this pathway.

From the above background, the appearance of a marker molecule for efficiently diagnosing melanoma and a new therapeutic target molecule that can be used for an effective therapeutic method is expected. This invention responds to the said request, and makes it a subject to contribute to development and improvement of the medical treatment (diagnosis method, treatment method) with respect to melanoma.

Our research group has been conducting research with the goal of identifying melanoma-specific marker molecules. As a result, four types of novel melanoma-related molecules (genes) that can be used for effective diagnosis and treatment of melanoma: PDZ domain containing ring inger 3 (PDZRN3), Deltex-3-like (DTX3L), and Chondroitin sulfate synthase 3 (CSS3), brain protein 44-like (BRP44L) was successfully identified. The levels of these four genes and their expression products (proteins) were significantly different between benign and malignant tumors. Further, as a result of detailed studies, it has been found that these molecules are deeply involved in the onset or progression (malignant transformation) of melanoma and the sensitivity and resistance to anticancer drugs and drugs having antitumor effects. Notably, PDZRN3, DTX3L, CSS3, and BRP44L significantly increased expression levels in both mouse melanoma without Braf mutation (RET-mouse and B16 strain) and human melanoma with BRAF mutation (PDZRN3, DTX3L). , CSS3) or decrease (BRP44L), it can be expected to be an effective target in the treatment of melanoma that is resistant to BRAF inhibitors. That is, these molecules that have been successfully identified can provide a solution to the problems of molecular targeted drugs used in current melanoma treatment.
The following invention is mainly based on the above results and considerations.
[1] A melanoma-specific biomarker comprising a biomolecule selected from the group consisting of PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene, CSS3 protein, BRP44L gene and BRP44L protein.
[2] Using the level in a sample of one or more biomolecules selected from the group consisting of PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene, CSS3 protein, BRP44L gene and BRP44L protein as an index Characteristic melanocyte tumor testing method.
[3] The melanocyte-based tumor examination method according to [2], comprising the following steps (1) to (3):
(1) a step of preparing a subject-derived specimen;
(2) detecting one or more of the biomolecules in the specimen; and (3) determining the malignancy of the melanocyte tumor based on the detection result.
[4] For the PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene and CSS3 protein, according to the criteria that the detection value is high and the malignancy is high, or that the detection possibility is high,
For the BRP44L gene and the BRP44L protein, the melanocyte according to [3], wherein the determination of step (3) is performed according to the criterion that the malignancy is high if the detection value is low, or the criterion that the malignancy is high if it cannot be detected. Tumor examination method.
[5] The melanocyte-based tumor examination method according to [3] or [4], wherein the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value of the control sample.
[6] The determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value in a sample collected in the past from the same subject, [3] or [ 4] The melanocyte tumor test method according to [4].
[7] The melanocyte tumor test method according to any one of [2] to [6], wherein the specimen is skin tissue, blood, plasma, serum, urine, saliva, sweat, or tumor tissue.
[8] A melanocyte tumor test reagent comprising a substance exhibiting specific binding property to the melanoma-specific biomarker according to [1].
[9] The melanocyte tumor test reagent according to [8], wherein the substance is an antibody.
[10] A melanocyte-based tumor test kit comprising the melanocyte-based tumor test reagent according to [8] or [9].
[11] A melanoma therapeutic agent comprising a compound that suppresses the expression of a target gene selected from the group consisting of a PDZRN3 gene, a DTX3L gene, and a CSS3 gene as an active ingredient.
[12] The melanoma therapeutic agent according to [11], wherein the compound is a compound selected from the group consisting of the following (a) to (e):
(a) siRNA targeting the target gene;
(b) a nucleic acid construct that generates siRNA targeting the target gene in the cell;
(c) a single-stranded RNA having an expression suppressing sequence that suppresses expression of the target gene and a complementary sequence that anneals to the sequence;
(d) an antisense nucleic acid that targets the transcript of the target gene;
(e) Ribozymes that target transcripts of target genes.
[13] A therapeutic agent for melanoma comprising the following (A) or (B) as an active ingredient and used in combination with an anticancer agent or a drug having an antitumor action:
(A) BRP44L protein or a part thereof (however, it has an effective effect on the anticancer drug sensitivity of melanoma cells);
(B) An expression vector carrying the BRP44L gene or a part thereof (however, it encodes a protein having an action effective for anticancer drug sensitivity of melanoma cells).
[14] The melanoma therapeutic agent according to any one of [11] to [13], which is applied to melanoma exhibiting resistance to a BRAF inhibitor.
[15] A melanoma treatment method comprising the step of administering the melanoma therapeutic agent according to any one of [11] to [14] to a patient with melanoma.

PDZRN3 gene expression level in melanoma model mouse tumors. PDZRN3 gene expression levels in benign tumors and malignant melanomas that spontaneously developed in melanoma model mice (RET-Tg) were measured using real-time PCR. PDZRN3 gene expression level is significantly increased in malignant melanoma compared to benign tumors. PDZRN3 gene expression level in human and mouse melanoma cell lines. a) Expression of PDZRN3 protein in human normal skin melanocytes (NHEM) and human melanoma cell lines (6 types). In all melanoma cell lines, the expression level of PDZRN3 protein is increased compared to NHEM. b) Expression level of Pdzrn3 protein in mouse melanoma cell lines (4 types). The B16 cell line is known to have lower invasive ability (metastatic activity) than other cell lines. The expression level in other cell lines with high invasive ability is greatly increased compared to B16 with low invasive ability. This suggests that Pdzrn3 may be associated with invasive ability. PDZRN3 protein expression level in human melanoma tissue (benign tumor: Benign, primary melanoma: Malignant, metastatic melanoma: Metastasis). After immunostaining with anti-PDZRN3 antibody, the expression level was measured by quantifying the staining level. The expression levels were categorized into three (negative / low, medium and high), and the ratio of each expression level was displayed as a band graph (bottom). Expression levels in primary and metastatic melanoma are significantly higher compared to benign tumors. Expression level of DTX3L gene in melanoma model mouse tumor. Real-time PCR (A), Western blotting (B) and immunohistochemical staining (DTX3L gene (A) and protein (B, C) expression levels in benign tumors and malignant melanomas spontaneously developed in melanoma model mice (RET-Tg) ( Measured using C). The expression levels of DTX3L gene and protein are greatly increased in malignant melanoma compared to benign tumors. Expression level of DTX3L protein in mouse and human melanoma cell lines. A) Expression level of Dtx3l protein in mouse melanoma cell lines (4 types). The B16 cell line is known to have lower invasive ability (metastatic activity) than other cell lines. B) Expression of DTX3L protein in human normal skin melanocytes (NHEM) and human melanoma cell lines (5 species). In all melanoma cell lines, the expression level of DTX3L protein is increased compared to NHEM. Expression level of DTX3L protein in human melanoma tissue (benign tumor: Benign, primary melanoma: primary melanoma, metastatic melanoma: Metastatic melanoma). After immunostaining with anti-DTX3L antibody, the expression level was measured by quantifying the staining level. The expression levels were categorized into three (negative / low, medium and high), and the ratio of each expression level was displayed as a band graph (bottom). Expression levels in primary and metastatic melanoma are significantly higher compared to benign tumors. Expression level of CSS3 gene in melanoma model mouse tumor. CSS3 has the same family genes, CSS1 and CSS2. The expression levels of these three genes in benign tumors and malignant melanomas spontaneously developed in melanoma model mice (RET-Tg) were measured using real-time PCR. CSS1 and CSS2 genes are both benign and malignant and their expression levels are low, but CSS3 gene expression is significantly increased in malignant melanoma. Expression level of CSS3 protein in human melanoma tissue (benign tumor: Benign, primary melanoma: Malignant, metastatic melanoma: Metastatic melanoma). After immunostaining with anti-CSS3 antibody, the expression level was measured by quantifying the staining level. The expression levels were categorized into three (negative / low, medium and high), and the ratio of each expression level was displayed as a band graph (bottom). Expression levels in primary and metastatic melanoma are significantly higher compared to benign tumors. Expression levels of BRP44L gene and protein in melanoma model mouse tumors. BRP44L gene (A) and protein (B) expression levels in benign tumors and malignant melanoma spontaneously developed in melanoma model mice (RET-Tg) were measured using real-time PCR (A) and Western blotting (B). BRP44L mRNA and protein are down-regulated in melanoma compared to benign tumors. Expression level of BRP44L gene in human melanoma tissue. The expression level of BRP44L mRNA in human melanoma tissue (benign tumor: nevus, melanoma: melanoma) was measured using real-time PCR. The expression level in human melanoma tissue is greatly reduced compared to benign tumors. Change of in vitro invasion ability (metastasis ability) by suppressing PDZRN3 expression. Using a shRNA forced expression vector, stable strains with reduced PDZRN3 expression were established in a mouse melanoma cell line (B16F10) (a), and the invasive ability of each stable strain was measured by an invasion assay (invasion assay) (b). Reduction in the expression level of PDZRN3 greatly inhibits the invasive ability inherent in melanoma cells. Changes in invasion ability (metastasis ability) in vivo by suppressing PDZRN3 expression. Using a shRNA forced expression vector, a stable strain in which PDZRN3 expression was reduced in a mouse melanoma cell line (B16F10) was established. In order to examine the invasive ability of each stable strain, the stable strain was injected into the tail vein of nude mice, and the subsequent metastatic activity to the lung was investigated (a). Reduction in the expression level of PDZRN3 greatly inhibits melanoma cell infiltration (metastasis) into the lung. On the other hand, the level of activation (phosphorylation) of invasive ability-related signal molecule (FAK) in each stable strain was investigated. All PDZRN3 expression-suppressed clones had decreased FAK phosphorylation (activity) levels (b) Changes in invitro invasion ability (metastasis ability) by suppressing DTX3L expression (mouse melanoma cell line). Establish stable strains with reduced DTX3L expression in mouse melanoma cell lines (B16F10) using shRNA forced expression vectors, and measure the infiltrating capacity of each stable strain by invasion assay (A) The activity of the signal molecule was measured (B). Decreasing the expression level of DTX3L suppresses the activity of Fak, Pi3k, and Akt signals, and greatly inhibits the invasion ability inherent in mouse melanoma cells. Change of in vitro invasion ability (metastasis ability) by suppressing DTX3L expression (human melanoma cell line). Establish stable strains with reduced expression of DTX3L in human melanoma cell line (G361) using shRNA forced expression vector, and measure the invasive ability of each stable strain by invasion assay (A) The activity of the signal molecule was measured (B). Decreasing the expression level of DTX3L suppresses the activities of Fak, Pi3k, and Akt signals, and greatly inhibits the invasive ability inherent to human melanoma cells. Change in in vivo invasion ability (metastasis ability) by suppressing DTX3L expression. In order to establish stable strains with reduced DTX3L expression in mouse melanoma cell line (B16F10) using shRNA forced expression vector, and to examine the invasive ability of each stable strain, stable strains were injected into the tail vein of nude mice, and then The lung metastasis activity was investigated. A) Since the injected cells express GFP, the lung metastasized cells can be visualized. B) Tissue section of a tumor derived from a lung cell line. C) Comparison of the number of tumors with lung metastases. Decreased expression level of DTX3L greatly inhibits infiltration (metastasis) of melanoma cells into the lung. Effect of DTX3L function inhibition on drug resistant melanoma cells. A human melanoma cell line (A375P) was exposed to the existing melanoma molecular target drug PLX4720 for a long period of time to create a resistant strain. When the expression of DTX3L was reduced by siRNA introduction (A), the effect on invasive ability was measured by an invasion assay (B). Suppressing DTX3L expression is also effective against PLX-resistant melanoma cells, and can solve the problems of existing molecular targeting drugs. Change in invasion ability (metastasis ability) in vivo by CSS3 forced expression. A stable strain in which CSS3 was forcibly expressed in a mouse melanoma cell line (B16F10) was established, and the invasion ability of each stable strain was measured by an invasion assay. Forced expression of CSS3 greatly activates the invasive ability inherent in melanoma cells. * P <0.05 Changes in invasion ability (metastasis ability) by suppressing CSS3 expression. a) Establish a stable strain with reduced CSS3 expression in a mouse melanoma cell line (B16F10 FLAG-CSS3) in which CSS3 was forcibly expressed using a shRNA forced expression vector, and measure the invasion ability of each stable strain by an invasion assay did. Decrease in the expression level of CSS3 inhibits the invasive ability inherent in melanoma cells. b) A CSS3 forced expression strain (+ CSS3) and an expression-suppressed stable strain (CSS3 sh) were injected into the tail vein of nude mice, and the subsequent metastatic activity to the lung was investigated. The number of metastases to the lung increased by CCS3 forced expression, and the number of metastases decreased by suppression of the expression. Change in response to anticancer drugs by forced expression of BRP44L. Cell death by treating human melanoma cell line SKMel28 (SK-GFP # 6) and stable strains forcibly expressing BRP44L (SK-B # 7, SK-B # 8) with one kind of etoposide as an anticancer agent And cell viability was measured. Under the high expression of BRP44L, cell death induction by etoposide was more strongly induced, so forced expression of BRP44L may increase the sensitivity to anticancer agents.

1. Melanoma-specific biomarker The first aspect of the present invention relates to a melanoma-specific biomarker. In the present specification, the “melanoma-specific biomarker” refers to a biomolecule that serves as an indicator of melanoma. As will be described later, the melanoma-specific biomarker is useful for determining whether or not it is a melanoma and for determining the malignancy of melanoma. The biomarker of the present invention is applied to, for example, a patient suffering from or possibly suffering from melanoma, or a sample / analyte derived from the patient.

In the present specification, “biomolecule” refers to a molecule (compound) found in the living body. In the present invention, a specific biomolecule is used as a biomarker. In its use (typically, application to a melanocyte-based tumor examination method), a biomolecule in a sample / specimen separated from a living body is used. Become.

The biomarker of the present invention comprises a biomolecule that has been correlated with the malignancy of melanocyte tumors (in other words, an increase or decrease in melanoma-specific expression level), and is used for evaluating the malignancy of melanocyte tumors. It is a useful indicator. The biomarker of the present invention consists of PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene, CSS3 protein, BRP44L gene or BRP44L protein. For convenience of explanation, unless otherwise mentioned, genes and proteins are comprehensively expressed using gene symbols (for example, “PDZRN3” represents both the PDZRN3 gene and the PDZRN3 protein collectively). In order to identify the molecules constituting the biomarker of the present invention, the sequences (gene sequences and amino acid sequences) of each molecule registered in a public database are shown below.
PDZRN3: SEQ ID NO: 1 (gene sequence, NCBI nucleotide database Accession No .: NM_015009), SEQ ID NO: 2 (amino acid sequence, NCBI protein database Accession No .: NP_055824)
DTX3L: SEQ ID NO: 3 (gene sequence, NCBI nucleotide database Accession No .: NM_138287), SEQ ID NO: 4 (amino acid sequence, NCBI protein database Accession No .: NP_612144)
CSS3: SEQ ID NO: 5 (gene sequence, NCBI nucleotide database Accession No .: NM_175856), SEQ ID NO: 6 (amino acid sequence, NCBI protein database Accession No .: NP_787052)
BRP44L: SEQ ID NO: 7 (gene sequence, NCBI nucleotide database Accession No .: NM_016098), SEQ ID NO: 8 (amino acid sequence, NCBI protein database Accession No .: NP_057182)

2. Melanoma Tumor Test Method The second aspect of the present invention relates to the use of the biomarker of the present invention, and provides a test method for melanocyte tumor (hereinafter also referred to as “test method of the present invention”). The test method of the present invention is useful as a means for determining the malignancy of a melanocyte tumor. Malignant melanoline tumors, that is, melanoma accounts for only about 5% of skin cancer, but it is highly metastatic and accounts for 80% of skin cancer deaths. For this reason, melanoma is said to be one of the most malignant cancers among skin cancers. The test method of the present invention provides useful information for diagnosing melanocyte tumors and contributes to the prevention and treatment of melanoma. According to the test method of the present invention, it is possible to easily and objectively determine the malignancy of a melanocyte tumor.

In the test method of the present invention, the level of the biomarker of the present invention in a specimen derived from a subject is used as an index. Here, “level” typically means “amount” or “concentration”. However, the term “level” is also used to indicate whether or not a molecule to be detected can be detected (ie, the presence or absence of an apparent presence) in accordance with common practice and common technical knowledge.

In the present invention, four types of biomarkers (PDZRN3, DTX3L, CSS3, BRP44L) can be used alone or in any combination. Different types of melanocyte tumors can be handled by using different biomarkers. That is, the present invention has an advantage that a wide range of cases can be dealt with.

Basically, the more types of biomarkers that are used together, the better the inspection accuracy and reliability. Therefore, preferably two or more types (for example, a combination of PDZRN3 and DTX3L, a combination of DTX3L and CSS3, a combination of CSS3 and BRP44L), more preferably three or more types are used in combination. In a particularly preferred embodiment, all of the above four types are used in combination. In contrast to other molecules (PDZRN3, DTX3L, CSS3), BRP44L showed a characteristic tendency that the expression level decreased in melanoma cells, as shown in Examples described later. In consideration of this fact, the use of BRP44L in combination with other biomarkers (one or more of PDZRN3, DTX3L, and CSS3) can be expected to have an effect of improving the accuracy of pathological diagnosis in a pathological specimen of a tumor. Note that the number and type of biomarkers to be employed may be determined in consideration of the required accuracy and the ease of inspection.

Typically, the inspection method of the present invention performs the following steps.
(1) A step of preparing a specimen derived from a subject (2) A step of detecting one or more biomolecules in the specimen (that is, one or more biomarkers employed as an index) (3) A step of determining the malignancy of the melanocyte tumor based on the detection result

In step (1), a specimen derived from the subject is prepared. As the specimen, skin tissue, blood, plasma, serum, urine, saliva, sweat, tumor tissue or the like of the subject can be used. The subject is not particularly limited. That is, the present invention can be widely applied to those who need to determine the malignancy of melanocyte tumors. “A person who needs to determine the malignancy of a melanocyte tumor” is typically a person who has developed a melanocyte tumor, but a subject who is suspected of developing a melanocyte tumor (potential patient) It is good. According to the test method of the present invention, the malignancy of a melanocyte tumor can be determined based on an objective index such as the level of a biomarker (biomolecule). The determination result is useful information for diagnosing melanocyte tumors, and is useful for determining a more appropriate treatment policy (selecting an effective treatment method, etc.). Therefore, the present invention contributes to the improvement of the therapeutic effect and the improvement of the patient's QOL (Quality of Life). In pathological diagnosis of melanocyte tumors, even a skilled laboratory technician or doctor may be at a loss. The highly objective data provided by the present invention provides information that is auxiliary but highly useful, especially in such cases.

In step (2), biomarkers in the specimen are detected. It is not essential to strictly quantify the level of the biomarker. That is, the level of the biomarker may be detected to the extent that the malignancy of the melanocyte tumor can be determined in the subsequent step (3). For example, detection can be performed so that it can be determined whether or not the level of the biomarker in the sample exceeds a predetermined reference value.

検出 Biomarker detection method is not particularly limited. When the biomarker adopted is a specific gene, it can be detected by quantifying the mRNA of the gene by a method using a nucleic acid amplification reaction such as RT-PCR. Nucleic acid amplification reactions include PCR (Polymerase reaction) method or its modified method, and LAMP (Loop-Mediated Isothermal Amplification) method (Tsugunori Notomi et al. Nucleic Acids Research, Vol.28, No.12, e63, 2000) ; Kentaro Nagamine, Keiko Watanabe et al. Clinical Chemistry, Vol.47, No.9, 1742-1743, 2001), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) (Patent No. 3433834) ), NASBA (Nucleic Acid Sequence-Based Amplification) method, LCR (Ligase Chain Reaction) method, 3SR (Self-sustained Sequence Replication) method, SDA (Standard Displacement Amplification) method, TMA (Transcription Medium Amplification) method, RCA ( Rolling Circle Amplification) can be used.

On the other hand, when the biomarker employed is a specific protein, the biomarker is preferably detected using an immunological technique. According to immunological techniques, rapid and sensitive detection is possible. Also, the operation is simple. In measurement by an immunological technique, a substance having specific binding property to the biomarker to be used is used. As the substance, an antibody is usually used, but any substance can be used as long as it has a specific binding property to the biomarker and can measure the binding amount. In addition, not only a commercially available antibody but the antibody newly prepared using the immunological method, the phage display method, the ribosome display method etc. may be used.

Examples of measurement methods include immunohistochemistry, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method), radioimmunoassay (RIA method), and Western blot method. According to the immunohistochemical staining method, the detection target can be detected quickly and with high sensitivity. Also, the operation is simple. Therefore, the burden on the subject (patient) accompanying the detection is reduced. In immunohistochemical staining of living tissues, generally, (1) fixation / paraffin embedding (or frozen embedding), (2) deparaffinization (not required for free embedding), (3) primary antibody reaction, ( 4) Add the labeling reagent, (5) color reaction, and (6) dehydration, penetration, and encapsulation. If necessary, prior to the primary antibody reaction, antigen activation treatment, endogenous peroxidase removal treatment (when peroxidase is used as a labeling substance), nonspecific reaction inhibition (treatment with bovine serum albumin solution, etc.) are performed. Is called. If necessary, nuclear staining (for example, Mayer's hematoxylin can be used) is performed before dehydration, penetration, and encapsulation. Various documents and books can be referred to for immunohistochemical staining of biological tissues (for example, “Enzyme Antibody Method, Revised 3rd Edition”, edited by Keiichi Watanabe and Kazuho Nakane, interdisciplinary project).

In step (3), the malignancy of the melanocyte tumor is determined based on the detection result. In order to enable accurate determination, it is preferable to perform the determination after comparing the detection value obtained in step (2) with the detection value of the control sample (control). For the control, for example, the biomarker level of healthy human skin tissue or the biomarker level of nevi (benign) can be used.

The determination of malignancy may be either qualitative or quantitative. It should be noted that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.

The biomarker of the present invention includes a biomarker (PDZRN3, DTX3L and CSS3) in which an increase in expression level correlates with malignancy of melanocyte tumor, and a biomarker (BRP44L) in which a decrease in expression level correlates with malignancy of melanocyte tumor )). Hereinafter, for convenience of explanation, the former is referred to as a first group of biomarkers, and the latter is referred to as a second group of biomarkers. Typically, the following criteria are employed for each biomarker. Needless to say, the criterion “high detection value is high malignancy” is synonymous with the criterion “low detection value is low malignancy” (the same applies to other criteria).

”Biomarkers that adopt the criteria of“ High detection value means high malignancy ”or“ Detection is high malignancy ”: Biomarkers of the first group (PDZRN3, DTX3L, CSS3)

”Biomarker that adopts the criterion of“ high malignancy when detection value is low ”or the criterion of“ high malignancy when detection is not possible ”: Biomarker of the second group (BRP44L)

∙ More specific determination is possible by setting a reference value for each biomarker. A specific example of the determination method when setting the reference value is shown below.

(First group biomarker determination example 1)
When the detected value of the biomarker (the level in the sample) is higher than the reference value, it is judged as “malignant” or “high possibility of malignancy”, and the detected value of the biomarker (the level in sample) is higher than the reference value Is low, it is judged as “benign” or “high possibility of benign”.

(Judgment example 2 of biomarker of the first group)
A reference value 1 for distinguishing benign and primary melanoma and a reference value 2 for distinguishing primary melanoma and metastatic melanoma are set (reference value 1 <reference value 2), and the determination is made as follows. It should be noted that a specific probability (for example, a determination of “80% or more probability”) may be shown instead of the evaluation “high possibility”.
When the detected value of the biomarker is smaller than the reference value 1: “benign” or “high possibility of benign”
When reference value 1 ≦ detection value of biomarker <reference value 2: “Primary melanoma” or “High possibility of primary melanoma”
Reference value 2 <detection value of biomarker: “metastatic melanoma” or “high possibility of metastatic melanoma”

(Judgment example 3 of biomarker of the first group)
A plurality of reference values that divide the malignancy level are set and determined as follows. In this example, four reference values (a <b <c <d) are set, but the number of reference values and the number of malignancy levels associated therewith are not limited to this.
If the detected value of the biomarker <a: Grade 1
When a ≦ biomarker detection value <b: Grade 2
When b ≦ detection value of biomarker <c: Grade 3
When c ≦ biomarker detection value <d: Grade 4
When d ≦ biomarker detection value: Grade 5

(Example 2 of determination of biomarkers in the second group)
When the detected value of the biomarker (level in the sample) is lower than the reference value, it is determined as “malignant” or “high possibility of malignancy”, and the detected value of the biomarker (level in the sample) is higher than the reference value Is determined to be “benign” or “high possibility of benign”.

(Second group biomarker determination example 2)
A reference value 1 for distinguishing benign and primary melanoma and a reference value 2 for distinguishing primary melanoma and metastatic melanoma are set (reference value 1 <reference value 2), and the determination is made as follows. It should be noted that a specific probability (for example, a determination of “80% or more probability”) may be shown instead of the evaluation “high possibility”.
When the detection value of the biomarker is smaller than the reference value 1: “It is a metastatic melanoma” or “It has a high possibility of a metastatic melanoma”
When reference value 1 ≦ detection value of biomarker <reference value 2: “Primary melanoma” or “High possibility of primary melanoma”
Reference value 2 <detection value of biomarker: “benign” or “high possibility of benign”

(Example 3 of determination of biomarkers of the second group)
A plurality of reference values that divide the malignancy level are set and determined as follows. In this example, four reference values (a <b <c <d) are set, but the number of reference values and the number of malignancy levels associated therewith are not limited to this.
Biomarker detection value <a: Grade 5
When a ≦ biomarker detection value <b: Grade 4
When b ≦ detection value of biomarker <c: Grade 3
When c ≦ biomarker detection value <d: Grade 2
If d ≤ biomarker detection value: Grade 1

When two or more types of biomarkers are used in combination, for example, after obtaining a determination result for each biomarker, all the determination results are comprehensively evaluated to obtain a final determination result. Alternatively, as shown in the following example (in this example, each of the

biomarkers

1 and 2 is the first group), by associating the combination of the detection values of the biomarkers with the determination result, A determination result may be obtained. In addition, also when combining 3 or more types of biomarkers, a determination result can be obtained according to the following examples.
When detection value of biomarker 1 <a, detection value of biomarker 2 <a ′: Grade 1
When a ≦ detection value of biomarker 1 <b, a ′ ≦ detection value of biomarker 2 <b ′, Grade 2
When b ≦ detection value of biomarker 1 <c, b ′ ≦ detection value of biomarker 2 <c ′ Grade 3
When c ≦ detection value of biomarker 1 <d and c ′ ≦ detection value of biomarker 2 <d ′ Grade 4
When d ≦ detection value of biomarker 1 and d ′ ≦ detection value of biomarker 2 Grade 5

When two or more types of biomarkers are used in combination, determination may be made as in the following (1) or (2).
(1) “malignant” or “malignant” when positive for all of the biomarkers included in the combination (greater than or equal to the cutoff value in the case of the first group biomarker and less than or equal to the cutoff value in the case of the second group biomarker) It is determined that “the possibility of malignancy is high”, and other cases are determined as “benign” or “high possibility of benign”.
(2) If any one of the biomarkers included in the combination is positive (in the case of the first group of biomarkers, the cut-off value or more, and in the case of the second group of biomarkers, the cut-off value or less), It is determined that “the possibility of malignancy is high”, and other cases are determined as “benign” or “high possibility of benign”.

Usually, diagnosis (detection) sensitivity and diagnosis (detection) specificity vary depending on the type and number of biomarkers to be combined. Therefore, an optimal combination of biomarkers may be selected according to the purpose. For example, combinations with high diagnostic sensitivity are suitable for screening tests. In contrast, a combination with a high diagnostic specificity is suitable for a test that requires a more reliable determination (for example, a secondary test or a tertiary test). By combining determination methods with different balances of diagnostic sensitivity and diagnostic specificity, it is possible to improve efficiency and improve accuracy or reliability. For example, after narrowing down using a combination of biomarkers giving high diagnostic sensitivity (primary test, screening test), a final determination is made using a combination of biomarkers giving high diagnostic specificity (secondary test). Not only such a two-step determination but also a three-step or higher determination can be performed.

The number of judgment categories, the level of the biomarker associated with each judgment category, and the judgment results can be arbitrarily set through preliminary experiments or the like without being bound by the above example. For example, the “reference value” when judging the grade of malignancy with a predetermined reference value (threshold) as the boundary, and the “biomarker level range” associated with the classification related to the grade of malignancy use a large number of specimens. Can be determined by statistical analysis.

In one embodiment of the present invention, the determination result is used to estimate the prognosis of melanoma. In this aspect, the prognosis of melanoma is estimated based on the determination result of step (3). Typically, it is estimated that the prognosis is bad (poor prognosis) in the case of a determination result that the malignancy is high, and the prognosis is good (good prognosis) in the case of the determination result that the malignancy is low. The prognostic level is set in advance corresponding to the malignancy level (for example, 1: very good, 2: good, 3: relatively good, 4: relatively bad, 5: bad, 6: very good. It is also possible to determine which level corresponds to a plurality of levels.

In one embodiment of the present invention, for the same subject, the level of a biomarker measured at a certain time point is compared with the level of a biomarker measured in the past, Or examine the degree of increase or decrease. The data regarding the change in the expression level of the biomarker obtained as a result is useful information for monitoring the malignancy, grasping the therapeutic effect, or estimating the prognosis. Specifically, for example, it can be determined that the grade of malignancy has increased or decreased or has not changed between the previous test and the current test based on the change in the biomarker level. If such evaluation is performed in parallel with the treatment of melanoma, not only can the therapeutic effect be confirmed, but also the signs of recurrence of melanoma can be grasped in advance. This makes it possible to determine a more appropriate treatment policy. Thus, the present invention can make a great contribution to maximizing the therapeutic effect and improving the patient's QOL (quality of life).

3. Test Reagents / Kits The present invention further provides reagents and kits for testing the malignancy of melanocyte tumors. The reagent of the present invention comprises a substance (hereinafter referred to as “binding molecule”) that exhibits specific binding to the biomarker of the present invention. Examples of binding molecules include antibodies, nucleic acid aptamers and peptide aptamers that specifically recognize biomarkers. The type and origin of the binding molecule are not particularly limited as long as it has specific binding properties for the biomarker employed. In the case of antibodies, any of polyclonal antibodies, oligoclonal antibodies (mixtures of several to several tens of antibodies), and monoclonal antibodies may be used. As a polyclonal antibody or an oligoclonal antibody, an anti-serum-derived IgG fraction obtained by animal immunization, or an affinity-purified antibody using an antigen can be used. Antibody fragments such as Fab, Fab ′, F (ab ′) ₂ , scFv, and dsFv antibodies may also be used.

The binding molecule may be prepared by a conventional method. If a commercial item is available, the commercial item may be used. For example, antibodies can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (biomarker or a part thereof) is prepared, and an animal such as a mouse, rat or rabbit is immunized using the antigen. An antigen can be obtained by purifying a biological sample. A recombinant antigen can also be used. A recombinant antigen can be obtained by, for example, introducing a gene encoding a biomarker (which may be a part of a gene) into a suitable host using a vector and expressing the gene in a recombinant cell obtained. Can be prepared.

In order to enhance the immunity-inducing action, an antigen to which a carrier protein is bound may be used. As the carrier protein, KLH (KeyholeHLimpet） Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen in which a biomarker (or part thereof) is expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

Immunization is repeated as necessary, and blood is collected when the antibody titer has sufficiently increased, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

On the other hand, monoclonal antibodies can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

Various modifications can be made to the obtained antibody on condition that the specific binding property to the biomarker is maintained. Such a modified antibody may be used as the reagent of the present invention.

If a labeled antibody is used as the specific binding molecule, it is possible to directly detect the amount of the bound antibody using the labeled amount as an index. Therefore, a simpler inspection method can be constructed. On the other hand, in addition to the necessity of preparing an antibody to which a labeling substance is bound, there is a problem that detection sensitivity is generally lowered. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having a specific binding property to an antibody specific to the biomarker employed. For example, when an antibody specific for a biomarker is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as a secondary antibody. Labeled secondary antibodies that can be used against various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd., Cosmo Bio Co., Ltd., etc.) and are appropriate for the reagents of the present invention. Can be appropriately selected and used.

Examples of labeling substances include peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-D-galactosidase, enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase, fluorescein isothiocyanate (FITC), Fluorescent materials such as tetramethylrhodamine isothiocyanate (TRITC) and europium, chemiluminescent materials such as luminol, isoluminol and acridinium derivatives, coenzymes such as NAD, biotin, and radioactive materials such as ¹³¹ I and ¹²⁵ I.

In one aspect, the reagent of the present invention is solid-phased according to its use. The insoluble support used for the solid phase is not particularly limited. For example, a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass can be used. The antibody can be supported on the insoluble support by physical adsorption or chemical adsorption.

The kit of the present invention contains the reagent of the present invention as a main component. Other reagents (buffers, blocking reagents, enzyme substrates, coloring reagents, etc.) and / or devices or instruments (containers, reaction devices, fluorescent readers, etc.) used in performing the test method may be included in the kit. Good. Moreover, it is preferable to include the biomarker molecule of the present invention or a fragment thereof as a standard sample in a kit. Usually, an instruction manual is attached to the kit of the present invention.

4). Melanoma treatment <Inhibition of target gene expression>
As a further aspect, the present invention provides a therapeutic agent for melanoma (hereinafter referred to as the medicament of the present invention). In one aspect, the medicament of the present invention comprises a compound that suppresses the expression of a target gene as an active ingredient. The target gene here is a PDZRN3 gene, a DTX3L gene, or a CSS3 gene. The compound that suppresses the expression of the target gene is a compound that suppresses the expression process of the target gene (including transcription, post-transcriptional regulation, translation, and post-translational regulation). Examples of the compound are as follows. In the present invention, “suppression of expression” may be either transient suppression or permanent suppression.
(a) siRNA targeting the target gene
(b) Nucleic acid construct that generates siRNA targeting the target gene in the cell (c) Single-stranded RNA having an expression suppressing sequence that suppresses the expression of the target gene and a complementary sequence that anneals to the sequence
(d) Antisense nucleic acid targeting the target gene transcript (e) Ribozyme targeting the target gene transcript

The above (a) and (b) are compounds used for expression suppression by so-called RNAi (RNA interference). In other words, according to the medicament of the present invention containing the compound (a) or (b) as an active ingredient, the expression of the target gene can be suppressed by RNAi. RNAi is a sequence-specific post-transcriptional gene repression process that can occur in eukaryotic cells. In RNAi for mammalian cells, a short double-stranded RNA (siRNA) having a sequence corresponding to the sequence of the target mRNA is used. Usually siRNAs are 21-23 base pairs. Mammalian cells are known to have two pathways (sequence specific pathway and sequence non-specific pathway) that are affected by double-stranded RNA (dsRNA). In sequence-specific pathways, relatively long dsRNAs are split into short interfering RNAs (ie siRNAs). On the other hand, a sequence non-specific pathway is considered to be caused by an arbitrary dsRNA regardless of the sequence as long as it has a predetermined length or more. In this pathway, dsRNA is activated by two enzymes, namely PKR, which stops all protein synthesis by phosphorylating the translation initiation factor eIF2, and 2 ', 5' oligoadenyl, which is involved in the synthesis of RNAase L activation molecules. Acid synthase is activated. In order to minimize the progression of this non-specific pathway, it is preferable to use double stranded RNA (siRNA) shorter than about 30 base pairs (Hunter et al. (1975) J Biol Chem 250: -17409-17 Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8 Wanna)

In order to generate target-specific RNAi, a siRNA composed of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto is introduced into the cell or expressed in the cell. Just do it. The above (a) is a compound corresponding to the former method, and similarly the above (b) is a compound corresponding to the latter method.

The siRNA targeting the target gene (in the present invention, the target gene) usually hybridizes with a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of its complementary sequence. Double stranded RNA. The length of the “continuous region” here is usually 15 to 30 bases, preferably 18 to 23 bases, more preferably 19 to 21 bases.

It is known that double-stranded RNA having an overhang of several bases at the end exhibits a high RNAi effect. Therefore, in the present invention, it is preferable to employ siRNA having such a structure. The length of the base that forms the overhang is not particularly limited, but is preferably 2 bases (for example, TT, UU).

SiRNA consisting of modified RNA may be used. Examples of modifications herein include phosphorothioation and the use of modified bases (eg, fluorescently labeled bases).

SiRNA can be designed and prepared by conventional methods. In designing siRNA, a sequence unique to a target sequence (continuous sequence) is usually used. Note that programs and algorithms for selecting an appropriate target sequence have been developed.

The “nucleic acid construct that generates siRNA in a cell” in (b) above refers to a nucleic acid molecule that, when introduced into a cell, produces a desired siRNA (siRNA that causes RNAi against a target gene) by a process in the cell. . One example of the nucleic acid construct is shRNA (short hairpin RNA). shRNA has a structure (hairpin structure) in which a sense RNA and an antisense RNA are linked via a loop structure part, and the loop structure part is cleaved in a cell to form a double-stranded siRNA, resulting in an RNAi effect. The length of the loop structure is not particularly limited, but is usually 3 to 23 bases.

Another example of a nucleic acid construct is a vector that can express a desired siRNA. Such vectors include those that express shRNA that is converted to siRNA by a later process (inserted with a sequence encoding shRNA) (referred to as stem loop type or short hairpin type), sense RNA and antisense RNA. Are vectors that are expressed separately (referred to as tandem type). Those skilled in the art can prepare these vectors according to conventional methods (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99: 1443-1448; Paul CP et al. (2002) Nature Biotechnology 19: 505-508; Sui G et.al. (2002) Proc Natl Acad Sci USA 99 (8): 5515-5520; addPaddison PJ et al. (2002) Genes Dev. 16: 948-958 etc.). Currently, various RNAi vectors are available. You may decide to construct | assemble the vector of this invention using such a well-known vector. In this case, after preparing an insert DNA encoding a desired RNA (for example, shRNA), it is inserted into a cloning site of the vector to obtain an RNAi expression vector.

It should be noted that the origin and structure of the vector are not limited as long as it has a function of generating siRNA that exerts RNAi action on the target gene in the cell. Accordingly, various viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors, etc.), non-viral vectors (liposomes, positively charged liposomes, etc.) and the like can be used. it can. Examples of promoters that can be used in the vector are U6 promoter, H1 promoter, and tRNA promoter. These promoters are RNA polymerase III type promoters, and high expression efficiency can be expected.

It has been reported that a single-stranded RNA having a predetermined structure is useful for suppressing the expression of a target gene (for example, WO2012 / 005368, JP2013-55913A, JP2013-138881A, JP2013-13A). No. 153736). Therefore, in one embodiment of the present invention, single-stranded RNA (above (c)) is used to suppress the expression of a target gene by the same mechanism as the suppression of expression by siRNA (that is, RNA interference). The single-stranded RNA of the present invention has an expression suppressing sequence corresponding to the target gene and a complementary sequence that can be annealed to the sequence. The order in which the expression suppressing sequence and the complementary sequence are linked is not particularly limited. Further, the expression suppressing sequence and the complementary sequence may be directly linked or may be linked via a linker region. The linker region can be composed of nucleotide residues or non-nucleotide residues (for example, comprising a structure such as polyalkylene glycol, pyrrolidine skeleton, piperidine skeleton, etc.). As an example of the form of the single-stranded RNA of the present invention, a molecule that forms a double-stranded structure (stem structure) by annealing a 5′-side region and a 3′-side region (Example 1), a 5′-side region, and a 3′-side region A molecule (Example 2) that forms two double-stranded structures (stem structures) by intramolecular annealing of the side regions separately can be given.

The expression-suppressing sequence is a sequence showing an activity of suppressing the expression of the target gene when the single-stranded RNA of the present invention is introduced into the cell. Typically, a sequence that causes expression suppression (ie, RNA interference) by siRNA is used as the expression suppression sequence. For example, the sequence of RNA constituting the above-described siRNA (above (a)) can be used as an expression suppressing sequence. The length of the expression suppressing sequence is not particularly limited, but is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19 to 21 bases long.

The length of the single-stranded RNA of the present invention is not particularly limited. The total number of bases constituting the single-stranded RNA (the total number of bases) has a lower limit of, for example, 38 bases, preferably 42 bases, more preferably 50 bases, still more preferably 51 bases, particularly preferably 52 bases. The upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, particularly preferably 80 bases. When the single-stranded RNA of the present invention includes a linker region, the total number of bases excluding the linker region is, for example, 38 bases, preferably 42 bases, more preferably 50 bases, more preferably 51 bases, particularly preferably the lower limit. Is 52 bases, and the upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, particularly preferably 80 bases.

In addition, when designing or preparing the single-stranded RNA of the present invention, it is possible to refer to past reports such as the above-mentioned patent publications.

The above (d) is a compound used for expression suppression by the antisense method. In other words, according to the medicament of the present invention containing the compound (d) as an active ingredient, the expression of the target gene can be suppressed by the antisense method. In the case of inhibiting expression by an antisense method, for example, an antisense construct that generates RNA complementary to a unique part of mRNA encoding a target gene when transcribed in the target cell is used. Such an antisense construct is introduced into a target cell, for example, in the form of an expression plasmid. On the other hand, as an antisense construct, an oligonucleotide probe that hybridizes with an mRNA / or genomic DNA sequence encoding a target gene and inhibits its expression when introduced into the target cell can also be employed. As such an oligonucleotide probe, one that is resistant to endogenous nucleases such as exonuclease and / or endonuclease is preferably used.

When a DNA molecule is used as the antisense nucleic acid, oligodeoxyribonucleotide derived from a region containing a translation initiation site (for example, a region of -10 to +10) of mRNA encoding the target gene is preferable.

It is preferable that the complementarity between the antisense nucleic acid and the target nucleic acid is exact, but some mismatch may exist. The ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids. Usually, the longer the antisense nucleic acid used, the more stable duplexes (or triplexes) can be formed with the target nucleic acid, even if the number of mismatches is large. One skilled in the art can ascertain the degree of acceptable mismatch using standard techniques.

The antisense nucleic acid may be DNA, RNA, a chimeric mixture thereof, or a derivative or modified type thereof. Moreover, it may be single-stranded or double-stranded. By modifying the base moiety, sugar moiety, or phosphate skeleton moiety, the stability, hybridization ability, etc. of the antisense nucleic acid can be improved. In addition, antisense nucleic acids can be used to promote cell membrane transport (for example, Letsingeretset al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; 556Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652; see PCT Publication No. W088 / 09810, published Dec 15, 15, 1988), or substances that enhance affinity for specific cells.

The antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems). For example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (1988), Proc. Natl. Acad. Sci. USA 85: 7448- 7451 etc. can be referred to.

Strong promoters such as pol II and pol III can be used to enhance the action of antisense nucleic acids in target cells. That is, when a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, a sufficient amount of the antisense nucleic acid can be transcribed by the action of the promoter.

The expression of the antisense nucleic acid can be performed by any promoter (inducible promoter or constitutive promoter) known to function in mammalian cells (preferably human cells). For example, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter derived from the 3 ′ end region of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes zoster thymidine A promoter such as a kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445) can be used.

In one embodiment of the present invention, expression suppression by ribozyme is used (in the case of the compound (e) above). The target mRNA can be destroyed using a ribozyme that cleaves the mRNA with a site-specific recognition sequence, but preferably a hammerhead ribozyme is used. For the construction method of the hammerhead ribozyme, for example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to.

As in the case of using the antisense method, for example, for the purpose of improving stability and target ability, a ribozyme may be constructed using a modified oligonucleotide. In order to generate an effective amount of ribozyme in a target cell, for example, a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a strong promoter (for example, pol II or pol III) can be used. preferable.

<Combination with anticancer agents>
As a result of the study by the present inventors, the fact that BRP44L is involved in resistance to anticancer agents has been clarified (examples described later). Based on this finding, in another embodiment of the medicament of the present invention, the sensitivity to an anticancer agent is increased by increasing the expression level of BRP44L in the target cells, thereby increasing the therapeutic effect of the anticancer agent. When treated with the medicament of this embodiment, the therapeutic results are improved as compared to treatment with the anticancer agent alone. “Improving therapeutic results” here includes an increase in therapeutic effect, an improvement in response rate or effectiveness rate, and a reduction or avoidance of side effects (at least one of which is achieved according to the present invention). ). Moreover, according to the medicine of this aspect, since the effect of the anticancer agent is enhanced, the amount of the anticancer agent used and the type of the anticancer agent (when two or more anticancer agents are used in combination) It is also possible to reduce this.

The pharmaceutical of this embodiment contains the following (A) (BRP44L protein) or (B) (expression vector holding the BRP44L gene) as an active ingredient, and is used in combination with an anticancer drug or a drug having an antitumor action.

(A) BPP44L protein In one embodiment of the present invention, BRP44L protein is an active ingredient. A part (fragment) of the BPP44L protein can also be used as an active ingredient as long as it exhibits an effective action on the anticancer drug sensitivity of melanoma cells. The amino acid sequence of human BRP44L protein is registered in a public database (SEQ ID NO: 8: NCBI protein database Accession No .: NP_057182). A polypeptide containing an amino acid sequence equivalent to the amino acid sequence can also be used as the BRP44L protein. The “equivalent amino acid sequence” here is partially different from the reference amino acid sequence (for example, the amino acid sequence of SEQ ID NO: 8), but the difference is related to the function of the protein (anticancer drug sensitivity of melanoma cells). It means an amino acid sequence that has no substantial effect on the effective action. Therefore, substantial functional identity is recognized between the reference amino acid sequence and the equivalent amino acid sequence. In order to determine the presence or absence of substantial functional identity, for example, using the experimental system (animal model or cell evaluation) described in the examples below, the effect on the anticancer drug sensitivity of melanoma cells What is necessary is just to confirm that there is no substantial difference between two amino acid sequences in terms of effect.

“Different in part of amino acid sequence” typically means deletion or substitution of 1 to several amino acids (upper limit is 3, 5, 7, 10) constituting the amino acid sequence. Alternatively, it means that a mutation (change) has occurred in the amino acid sequence due to addition, insertion, or a combination of 1 to several amino acids (the upper limit is, for example, 3, 5, 7, 10). Differences in amino acid sequences here are allowed as long as there is no significant decrease in the above functions. As long as this condition is satisfied, the positions where the amino acid sequences are different are not particularly limited, and differences may occur at a plurality of positions. The term “plurality” as used herein refers to, for example, a number corresponding to less than about 30% of all amino acids, preferably a number corresponding to less than about 20%, and more preferably a number corresponding to less than about 10%. The number is preferably less than about 5%, and most preferably less than about 1%. That is, the equivalent amino acid sequence is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, still more preferably about 95% or more, and most preferably about 99% or more with the reference amino acid sequence. Has sequence identity.

It is preferable that the difference between the reference amino acid sequence and the equivalent amino acid sequence is caused by a conservative amino acid substituent. As used herein, “conservative amino acid substitution” refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). A conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.

Incidentally, the sequence identity (%) of two amino acid sequences or two nucleic acid sequences (hereinafter, “two sequences” is used as a term including them) can be determined, for example, by the following procedure. First, two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is the same. Sequence identity is a function of the number of identical positions common to the two sequences (ie, sequence identity (%) = number of identical positions / total number of positions × 100), preferably for alignment optimization Take into account the number and size of gaps required.

比較 Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. Specific examples of mathematical algorithms that can be used for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68, and Karlin and Altschul (1993) Proc. Natl. There is a modified algorithm in Acad. Sci. USA 90: 5873-77, but it is not limited to this. Such an algorithm is incorporated in the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search may be carried out with the score = s100 and wordlength = 12 in the NBLAST program. In order to obtain an amino acid sequence equivalent to the reference amino acid sequence, for example, a BLAST polypeptide search may be performed using the XBLAST program with score = 50 and wordlength = 3. In order to obtain a gap alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. For details, refer to the NCBI web page. Examples of other mathematical algorithms that can be used for sequence comparison include those described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is incorporated in the ALIGN program available on, for example, the GENESTREAM network server (IGH （Montpellier, France) or the ISREC server. When using the ALIGN program for comparison of amino acid sequences, for example, a PAM120 residue mass table can be used, with a gap length penalty = 12 and a gap penalty = 4.

The identity of two amino acid sequences, using the Gloss program in the GCG software package, using a Blossom 62 matrix or PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3 Or 4 can be determined. Also, the identity of two nucleic acid sequences can be determined using the GAP program of the GCG software package, with gap weight = 50 and gap length weight = 3.

BRP44L can be easily prepared by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. it can. For example, it can be prepared by transforming a suitable host cell (for example, E. coli, yeast) with DNA encoding BRP44L and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, various modifications are possible if BRP44L is obtained as a recombinant protein. For example, if a DNA encoding BRP44L and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, BRP44L consisting of a recombinant protein linked to any peptide or protein Can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

From the viewpoint of qualitative uniformity and purity, it is preferable to prepare BRP44L by genetic engineering techniques. However, the method for preparing BRP44L is not limited to the genetic engineering method. For example, BRP44L can also be prepared from natural materials by standard techniques (crushing, extraction, purification, etc.).

(B) Expression vector holding BRP44L gene In one embodiment of the present invention, an expression vector holding the BRP44L gene is used as an active ingredient. As long as it encodes a protein that exhibits an effective action on the anticancer drug sensitivity of melanoma cells, a part thereof may be used instead of the BRP44L gene. “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell. In the expression vector according to the present invention, the BRP44L gene is retained so that it can be expressed. The type of vector is not particularly limited as long as the BRP44L gene can be introduced into the target cell and expressed in the target cell. The “vector” herein includes viral vectors and non-viral vectors. The gene transfer method using a virus vector skillfully utilizes the phenomenon that a virus infects cells, and high gene transfer efficiency can be obtained. Adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors and the like have been developed as virus vectors.

Non-viral vectors include liposomes, positively charged liposomes (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417, 1987), HVJ (Hemagglutinating virus of Japan) -Liposome (Dzau, VJ, Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci., 93: 11421-11425, 1996, Kaneda, Y., Saeki, Y. & Morishita , R., Molecular Med. Today, 5: 298-303, 1999). The expression vector in the present invention may be constructed as such a non-viral vector. Further, a YAC vector, a BAC vector or the like may be used.

In adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, a foreign gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Retroviral vectors are not suitable for gene transfer into non-dividing cells because cell division is required for integration of the viral genome into the host chromosome. On the other hand, lentivirus vectors and adeno-associated virus vectors cause integration of foreign genes into the host chromosome after infection even in non-dividing cells. Therefore, these vectors are effective for stably and long-term expressing foreign genes in non-dividing cells.

Each virus vector can be prepared according to a previously reported method or using a commercially available dedicated kit. For example, an adenovirus vector can be prepared by the COS-TPC method or full-length DNA introduction method. The COS-TPC method is a homologous recombination that occurs in 293 cells by co-transfecting a recombinant cosmid incorporating the target cDNA or expression cassette and a parent virus DNA-terminal protein complex (DNA-TPC) into 293 cells. (Mayake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). On the other hand, the full-length DNA introduction method is a method for producing a recombinant adenovirus by subjecting a recombinant cosmid inserted with a target gene to restriction digestion and then transfecting 293 cells (Miho Terashima, Koki Kondo). Hiromi Kanegae, Izumi Saito (2003) Experimental Medicine 21 (7) 931.). The COS-TPC method can be performed using Adenovirus® Expression® Vector® Kit® (Dual® Version) (Takara Bio Inc.) and Adenovirus® genome® DNA-TPC (Takara Bio Inc.). In addition, the full-length DNA introduction method can be performed using Adenovirus® Expression® Vector® Kit® (Dual® Version) (Takara Bio Inc.).

On the other hand, retroviral vectors can be prepared by the following procedure. First, the virus genome (gag, pol, env gene) other than the packaging signal sequence between the LTRs (Long Terminal Repeat) existing at both ends of the virus genome is removed, and the target gene is inserted therein. The viral DNA thus constructed is introduced into a packaging cell that constitutively expresses the gag, pol, and env genes. Thereby, only the vector RNA having the packaging signal sequence is incorporated into the viral particle, and a retroviral vector is produced.

Developed by improving or specificizing fiber protein by modifying adeno vectors (specific infection vectors) and gutted vectors (helper-dependent vectors) that can be expected to improve the expression efficiency of target genes. Has been. The expression vector of the present invention may be constructed as such a viral vector.

The BRP44L gene inserted into the expression vector is preferably composed of a base sequence described in SEQ ID NO: 7 (NCBI nucleotide database Accession No .: NM_016098. However, a DNA having a base sequence equivalent to the base sequence (hereinafter "equivalent" (Referred to as “DNA”) can also be used as the BRP44L gene. The “equivalent base sequence” here is partly different from the reference base sequence (SEQ ID NO: 7), but it is encoded by the difference. A base sequence in which the function of the protein (effective effect on the sensitivity of melanoma cells to anticancer drugs) is not substantially affected.The specific example of equivalent DNA is complementary to the reference base sequence. A DNA that hybridizes under stringent conditions to a nucleotide sequence, where “stringent conditions” form a so-called specific hybrid that is non-specific. Such stringent conditions are known to those skilled in the art, such as Molecular 例えば Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M Ausubel et al., 1987) As stringent conditions, for example, hybridization solution (50% formamide, 10 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)), incubated at about 42 ° C. to about 50 ° C., then 0.1 × SSC And washing with 0.1% SDS at about 65 ° C. to about 70 ° C. More preferred stringent conditions include, for example, 50 hybridization solutions. Formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5) ) Can be mentioned.

As another specific example of equivalent DNA, it consists of a base sequence that includes substitution, deletion, insertion, addition, or inversion of one or more bases relative to a reference base sequence, and is effective in anticancer drug sensitivity of melanoma cells. Examples thereof include DNA encoding a protein that exhibits an effective action on the protein. Base substitution or deletion may occur at a plurality of sites. The term “plurality” as used herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it depends on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA It is. Such equivalent DNAs include, for example, restriction enzyme treatment, treatment with exonuclease and DNA ligase, position-directed mutagenesis (MolecularMCloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) Includes substitutions, deletions, insertions, additions, and / or inversions of bases using mutation introduction methods (Molecular Cloning, ingThird Edition, Chapterhap13, Cold Spring Harbor Laboratory Press, New York) Thus, it can obtain by modifying DNA which has a standard base sequence. The equivalent DNA can also be obtained by other methods such as ultraviolet irradiation.

As yet another example of equivalent DNA, there can be mentioned DNA in which a base difference as described above is recognized due to a polymorphism represented by SNP (single nucleotide polymorphism).

The BRP44L gene can be prepared by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. For example, the BRP44L gene can be isolated (and amplified) from a human cDNA library by appropriately using an oligonucleotide probe / primer that can specifically hybridize to the BRP44L gene. As the oligonucleotide probe primer, for example, DNA complementary to the base sequence shown in SEQ ID NO: 7 or a continuous part thereof is used. Oligonucleotide probes and primers can be easily synthesized using a commercially available automated DNA synthesizer. For the method of preparing a library used for preparing the BRP44L gene, for example, Molecular® Cloning, • Third • Edition, • Cold®Spring®Harbor®Laboratory®Press, and “New York” are helpful.

An equivalent DNA can be prepared by using a cDNA library derived from a non-human mammalian cell (for example, monkey, mouse, rat, pig, bovine) instead of the human cDNA library. As an example of the BRP44L gene derived from mammals other than humans, the sequence of the mouse-derived Brp44l gene is shown in SEQ ID NO: 15.

Examples of “anticancer agents” or “agents having antitumor activity” used in combination with the therapeutic agent of the present invention include platinum preparations such as cisplatin, carboplatin, and oxaliplatin, cyclophosphamide, fluorouracil (5-FU). Etoposide, doxorubicin, bleomycin, mitomycin, staurosporine.

<Application to melanoma resistant to BRAF inhibitor>
As a result of the study by the present inventors, PDZRN3, DTX3L, CSS3 and BRP44L significantly increased (PDZRN3, DTX3L, CSS3) or decreased the expression level in both mouse melanoma without Braf mutation and human melanoma with BRAF mutation. (BRP44L) was observed (Examples described later). Therefore, it can be said that PDZRN3, DTX3L, CSS3 and BRP44L are useful as target molecules in the treatment of melanoma regardless of the presence or absence of BRAF mutation. In fact, DTX3L became an effective target in melanoma cells (having a BRAF mutation) that acquired resistance to the BRAF inhibitor PLX (Examples described later). Thus, in a preferred embodiment, the medicament of the present invention is applied to melanomas that are resistant to BRAF inhibitors. Typically, the medicament of this embodiment is intended for treatment of melanoma patients who have acquired resistance to a BRAF inhibitor as a result of treatment with the BRAF inhibitor. However, patients with melanoma that are resistant to BRAF inhibitors because of their intrinsic characteristics or other causes than the use of BRAF inhibitors (for example, mutations associated with the development of symptoms or mutations due to the effects of other drugs) Can also be treated. Examples of BRAF inhibitors are PLX4032 and PLX4720.

In a drug applied to a melanoma resistant to a BRAF inhibitor, preferably, DTX3L is a target molecule. Therefore, as a preferred embodiment, a medicament comprising a compound that suppresses the expression of the DTX3L gene as an active ingredient is provided.

<Pharmaceutical formulation, administration, treatment target>
The pharmaceutical preparation of the present invention can be prepared according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories. The medicament of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (ie, a therapeutically effective amount). The amount of the active ingredient in the medicament of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1 wt% to about 95 wt% so as to achieve a desired dose. The medicament of the present invention is administered to the subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. Applied. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). When a nucleic acid construct is an active ingredient (for example, an embodiment using RNAi), not only in vivo administration but also ex vivo administration can be employed.

The “subject” to which the medicament of the present invention is administered is not particularly limited, and includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows) Pigs, goats, sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the medicament of the present invention is applied to humans. Although the dosage varies depending on the patient's symptoms, age, sex, weight, etc., those skilled in the art can appropriately set an appropriate dosage. In setting the administration schedule, it is possible to consider the patient's symptoms and the duration of the effect of the medicine.

As is apparent from the above description, the present application also provides a method for treating melanoma, which comprises administering a therapeutically effective amount of the medicament of the present invention to a melanoma patient.

The following studies were conducted in order to find melanoma-specific biomolecules (biomarkers and therapeutic targets).

1. Materials and Methods (1) Cells and mice Normal human skin melanocytes (NHEM) were purchased from Kurashiki Boseki Co., Ltd. (KURABO, Japan). Human melanoma cell lines SK-Mel28 and G361 are from RIKEN BioResource Center, human melanoma cell line MNT1 is from Dr. VJ Hearing (National Cancer Institute, NIH, Bethesda, MD), human melanoma cell lines A375P and A375M are from Dr. Dorothy C Bennett ( Provided by St George's, UK). Mouse melanoma cell lines (B16, B16F1, B16F10, B16BL6) were provided by Tohoku University Cell Bank. 304 / B6 mice (RET-mice) (Kato, M. et al., J Invest Dermatol. 111: 640-4 .; Iwamoto, T. et al., EMBO J. 10: 3167-75 .; Kato, M et al., Oncogene. 17: 1885-8, etc.) was used to collect melanocyte benign tumors and melanoma.

(2) Real-time PCR
Total RNA was extracted from cultured cells and tissues using High Pure RNA Kit (Roche Diagnostics), and cDNA was synthesized using Super-criptTM III reverse transcriptase (Invitrogen). Real-time RT-PCR was performed using a reagent of power SYBR1 Green PCR master mix (Applied Biosystems) and ABI Prism7500 sequence detection system (Applied Biosystems).

(3) Establishment of gene forced expression cell line and knockdown cell line The pCMV-c-Fa-Puro3 vector (Invitrogen) was used for the production of the gene forced expression cell line. After introducing the cDNA of the protein coding region into the pCMV-c-Fa-Puro3 vector, the cultured cells were transfected, and the cells were selected with 1 mg / ml puromycin (Wako Pure Chemical Industries, Ltd.). The pRNAT-U6-1-Neo vector (Invitrogen) was used for establishment of the knockdown cell line. After introducing the shRNA sequence into the pRNAT-U6-1-Neo vector, the cultured cells were transfected, and cells were selected with 1 mg / ml neomycin (Wako Pure Chemical Industries, Ltd.).

(4) In vitro and in vivo analysis of melanoma invasion activity In in vitro analysis, cell culture insert (FALCON) was coated with Matrigel (Corning), seeded with cells cultured under 1% FBS conditions, and cultured with 10% FBS After adding the liquid to the 24-well dish, the cell culture insert was set. After 24-48 hours, the cell culture insert was fixed in formalin, and the cells were stained with crystal violet. In in vivo analysis, cells cultured under 1% FBS conditions were injected from the tail vein of nude mice, dissected after 14 days, and examined for metastasis to the lung.

(5) Authorization This study was conducted at the Animal Experiment Committee (Nagoya University authorization number 26317, Chubu University authorization number 2410062), Recombinant DNA Experiment Committee (Nagoya University authorization number 13-76) Approved by Chubu University Approval Number 12-03) and Ethics Committee (Nagoya University Approval Number 2013-0070, Chubu University Approval Number 250007).

(6) Gene sequence and protein sequence The sequence of each gene and protein registered in a public database is described below.
Human PDZRN3 gene: SEQ ID NO: 1 (NCBI nucleotide database Accession No .: NM_015009)
Human PDZRN3 protein: SEQ ID NO: 2 (NCBI protein database Accession No .: NP_055824)
Human DTX3L gene: SEQ ID NO: 3 (NCBI nucleotide database Accession No .: NM_138287)
Human DTX3L protein: SEQ ID NO: 4 (NCBI protein database Accession No .: NP_612144)
Human CSS3 gene: SEQ ID NO: 5 (NCBI nucleotide database Accession No .: NM_175856)
Human CSS3 protein: SEQ ID NO: 6 (NCBI protein database Accession No .: NP_787052)
Human BRP44L gene: SEQ ID NO: 7 (NCBI nucleotide database Accession No .: NM_016098)
Human BRP44L protein: SEQ ID NO: 8 (NCBI protein database Accession No .: NP_057182)

Mouse Pdzrn3 gene: SEQ ID NO: 9 (NCBI nucleotide database Accession No .: NM_018884)
Mouse Pdzrn3 protein: SEQ ID NO: 10 (NCBI protein database Accession No .: NP_061372)
Mouse Dtx3l gene: SEQ ID NO: 11 (NCBI nucleotide database Accession No .: NM_001013371)
Mouse Dtx3l protein: SEQ ID NO: 12 (NCBI protein database Accession No .: NP_001013389)
Mouse Css3 gene: SEQ ID NO: 13 (NCBI nucleotide database Accession No .: NM_001081328)
Mouse Css3 protein: SEQ ID NO: 14 (NCBI protein database Accession No .: NP_001074797)
Mouse Brp44l gene: SEQ ID NO: 15 (NCBI nucleotide database Accession No .: NM_018819)
Mouse Brp44l protein: SEQ ID NO: 16 (NCBI protein database Accession No .: NP_061289)

2. Result: mRNA was purified from spontaneously occurring melanocyte benign tumors and melanomas in melanoma model mice (RET-Tg), and gene expression analysis was performed using DNA microarrays. Genes with stronger or weaker expression in melanoma compared to benign tumors Thus, PDZ domain containing ring finger 3 (PDZRN3), Deltex-3-like (DTX3L), Chondroitin sulfate synthase 3 (CSS3), and BRP44L gene were identified as melanoma-related genes.

(1) Gene / protein expression analysis
(a) PDZRN3
When the expression level of PDZRN3 gene in benign tumor and malignant melanoma spontaneously developed in melanoma model mice (RET-Tg) was measured using real-time PCR, PDZRN3 gene was compared with the expression level in benign tumors. The expression level was greatly increased (FIG. 1). When the expression of PDZRN3 protein in human normal skin melanocytes (NHEM) and human melanoma cell lines (6 types) was investigated, the expression level of PDZRN3 protein was increased in all melanoma cell lines compared to NHEM (Fig. 2a). In addition, the expression level of Pdzrn3 protein in mouse melanoma cell lines (4 types) was investigated. The B16 cell line is known to have lower invasive ability (metastatic activity) than other cell lines. Compared with B16 with low invasive ability, the expression level in other cell lines with high invasive ability is greatly increased (FIG. 2b), suggesting that Pdzrn3 may be associated with invasive ability.

The expression level of PDZRN3 protein in human melanoma tissues (benign tumor: Benign, primary melanoma: Malignant, metastatic melanoma: Metastasis) was measured by quantifying the staining level after immunostaining with anti-PDZRN3 antibody. The expression levels were categorized into 3 (negative / low, moderate, high) and the ratio of each expression level was compared. The expression levels in primary and metastatic melanoma were significantly higher than those in benign tumors. It became clear (Fig. 3).

(b) DTX3L
DTX3L gene and protein expression levels in benign tumors and malignant melanoma spontaneously developed in RET-Tg mice were measured using real-time PCR (FIG. 4A), Western blotting (FIG. 4B) and immunohistochemical staining (FIG. 4C). The expression level of DTX3L gene and protein was significantly increased in malignant melanoma compared to the expression level in benign tumors. The expression level of Dtx3l protein in mouse melanoma cell lines (4 types) was measured using Western blotting (FIG. 5A). The B16 cell line is known to have lower invasive ability (metastatic activity) than other cell lines. Compared to B16, which has low invasive ability, the expression level in other cell lines with high invasive ability is greatly increased, suggesting that Dtx3l may be associated with invasive ability. In addition, the expression level of DTX3L protein in human normal skin melanocytes (NHEM) and human melanoma cell lines (5 types) was measured using Western blotting (FIG. 5B). In all human melanoma cell lines, the expression level of DTX3L protein was increased compared to NHEM. The expression level of DTX3L protein in human melanoma tissues (benign tumor: Benign, primary melanoma: metastatic melanoma) was measured by quantifying the staining level after immunostaining with anti-DTX3L antibody. As a result of categorizing the expression level into 3 (negative / low, moderate, high) and comparing the ratio of each expression level, the expression level in primary melanoma and metastatic melanoma is significantly higher than in benign tumors It became clear (FIG. 6A, B).

(c) CSS3
CSS3 has the same family genes, CSS1 and CSS2. As a result of measuring the expression levels of these three genes in benign tumors and malignant melanomas spontaneously developed in melanoma model mice (RET-Tg) using real-time PCR, the expression levels of CSS1 and CSS2 genes are both benign and malignant and low. However, the expression level of CSS3 gene was significantly increased in malignant melanoma (FIG. 7). The expression level of CSS3 protein in human melanoma tissue (benign tumor: Benign, primary melanoma: Malignant, metastatic melanoma: Meta) was measured by quantifying the staining level after immunostaining with anti-CSS3 antibody (FIG. 8). ). As a result of categorizing the expression level into 3 (negative / low, moderate, high) and comparing the ratio of each expression level, the expression level in primary melanoma and metastatic melanoma is significantly higher than in benign tumors It became clear.

(d) BRP44L
BRP44L gene and protein expression levels in benign tumors and malignant melanomas spontaneously developed in melanoma model mice (RET-Tg) were measured using real-time PCR (FIG. 9A) and Western blotting (FIG. 9B). BRP44L mRNA and protein had decreased expression in melanoma compared to benign tumors. The expression level of BRP44L mRNA in human melanoma tissue (benign tumor: nevus, melanoma: melanoma) was measured using real-time PCR. As a result, the expression level in human melanoma tissue was significantly reduced compared to that in benign tumor. (Fig. 10).

(2) Functional analysis of each gene
(a) PDZRN3
Using a shRNA forced expression vector, stable strains with reduced expression of PDZRN3 in a mouse melanoma cell line (B16F10) were established (FIG. 11a), and the invasive ability of each stable strain was measured by an invasion assay (FIG. 11b). Cells with reduced expression of PDZRN3 have a greatly reduced number of infiltrating cells (cells stained in purple in the original image of FIG. 11b) (P1, P2 in FIG. 11b), and have a greater invasive ability inherent to melanoma cells. It was inhibiting. A stable strain with reduced expression of PDZRN3 was injected into the tail vein of nude mice, and the subsequent metastatic activity to the lung was investigated (FIG. 12a). The cell line (shPdzrn3) in which the expression level of PDZRN3 was reduced was greatly inhibited from infiltrating into the lung (metastasis) compared to the control line (shControl). When the activation (phosphorylation) level of signal molecule (FAK) related to invasion ability in each stable strain was investigated, the phosphorylation (activity) level of FAK decreased in all PDZRN3 expression-suppressed clones (P1 to P3). (FIG. 12b), it was suggested that PDZRN3 may control the activity of invasive ability-related signal molecule (FAK).

(b) DTX3L
Using a shRNA forced expression vector, stable strains with reduced expression of Dtx3l in a mouse melanoma cell line (B16F10) were established, and the invasion ability of each stable strain was measured by an invasion assay (FIG. 13A) and involved in invasion. The activity of intracellular signal molecules was measured (FIG. 13B). It was revealed that the decrease in the expression level of Dtx3l suppressed the activity of Fak, Pi3k, and Akt signals and greatly inhibited the invasion ability inherent in mouse melanoma cells. On the other hand, using the shRNA forced expression vector, stable strains with reduced expression of DTX3L were established in the human melanoma cell line (G361), and the invasive ability of each stable strain was measured by an invasion assay (FIG. 14A). The activity of intracellular signal molecules involved was measured (FIG. 14B). Both cell invasive ability and intracellular signal molecules involved in invasion are suppressed, and DTX3L controls melanoma invasion (metastasis) activity not only in mice but also in humans Became clear. Next, a stable strain in which DTX3L expression was reduced in a mouse melanoma cell line (B16F10) was established using an shRNA forced expression vector. In order to examine the invasive ability of each stable strain, the stable strain was injected into the tail vein of nude mice, and the subsequent metastatic activity to the lung was investigated. Since the injected cells express GFP, lung metastasized cells can be visualized (FIG. 15A). The decrease in the expression level of DTX3L significantly inhibited infiltration (metastasis) of melanoma cells into the lung (FIG. 15). Recently developed molecular therapeutics for BRAF, PLX4032 and PLX4720, are one of the new therapeutic candidates. However, long-term treatment with melanoma produces resistant cells and recurs frequently. In this experiment, the human melanoma cell line (A375P) was exposed to the melanoma molecule targeted drug PLX4720 for a long period of time to create a resistant strain, and the effect on the invasive potential when DTX3L expression was reduced by siRNA introduction (FIG. 16A). Measured by invasion assay (Figure 16B). Suppressing the expression of DTX3L is also effective against PLX-resistant melanoma cells, and a therapeutic effect on drug-resistant melanoma produced by existing molecular target reagents can be expected.

(c) CSS3
A stable strain in which CSS3 was forcibly expressed in a mouse melanoma cell line (B16F10) was established, and the invasive ability of each stable strain was measured by an invasion assay (FIG. 17). It was revealed that forced expression of CSS3 further activated the invasive ability inherent in melanoma cells. Using a shRNA forced expression vector, a stable strain with reduced CSS3 expression was established in a mouse melanoma cell line (B16F10 FLAG-CSS3) in which CSS3 was forcibly expressed, and the invasion ability of each stable strain was measured by an invasion assay ( FIG. 18a). Decrease in the expression level of CSS3 inhibited the invasive ability of melanoma cells. B16F10 control cells, CSS3 forced expression strain (+ CSS3), and expression-suppressed stable strain (CSS3 sh) were injected into the tail vein of nude mice, and then examined for metastatic activity to the lung. It was revealed that the number of metastases increased and the number of metastases decreased by suppressing the expression (FIG. 18b).

(d) BRP44L
Cell death by treatment of human melanoma cell line SKMel28 (SK-GFP # 6) and stable strains forcibly expressing BRP44L (SK-B # 7, SK-B # 8) with one kind of etoposide anticancer agent And cell viability was measured (FIG. 19). Under the high expression of BRP44L, cell death induction by etoposide was more strongly induced, suggesting that forced expression of BRP44L may increase the sensitivity to anticancer drugs.

3. Discussion All four types of genes and gene products (proteins) analyzed this time have large differences in their expression levels between benign tumors and malignant tumors (see Figs. 1 to 10), and their functions are also related to actual melanoma development. Or closely related to sensitivity to anti-cancer drugs (see FIGS. 11-19). If these molecules are used as biomarkers (diagnostic markers), not only can the accuracy of pathological diagnosis of melanocyte tumors, which are often difficult to diagnose, be improved, but also examination / diagnosis considering the level of malignancy becomes possible. The combined use of these biomarkers provides detailed and useful information in both early diagnosis and pathological diagnosis of melanoma. Since BRP44L had decreased expression in melanoma as opposed to the other 3 genes, the information obtained when BRP44L was used in combination with other biomarkers (one or more of PDZRN3, DTX3L, CSS3) It is particularly useful.

The four genes and proteins analyzed this time can inhibit melanoma activity very sensitively by suppressing or enhancing their expression (see FIGS. 11 to 19). PDZRN3, DTX3L, and CSS3 are mainly involved in invasion and metastasis, and BRP44L is involved in anticancer drug-induced cell death sensitivity. By using these molecules according to the melanoma characteristics of each patient, it may be possible to deal with various types of melanoma. Recently developed molecular therapeutics for BRAF, PLX4032 and PLX4720, are candidates for melanoma treatment. However, long-term treatment with melanoma produces resistant cells, and melanoma recurs frequently. In this experiment, we succeeded in significantly suppressing the metastasis of PLX4720-resistant cells by suppressing the expression of the DTX3L gene (FIG. 16). In other words, the newly identified melanoma-related molecule (a candidate molecule for molecular target therapy for melanoma) can be said to be effective for the problems of melanoma therapeutic agents such as PLX4032 and PLX4720.

New melanoma-related molecules are deeply involved in melanoma metastasis and sensitivity to anticancer drugs. The melanoma-related molecule has a particularly poor prognosis among various carcinomas, and can be expected to make a great contribution to the diagnosis and treatment of melanoma, which requires early detection for effective treatment.

The biomarker of the present invention is useful for early detection of melanoma, determination of malignancy of melanoma, and the like. In addition, biomolecules constituting biomarkers are deeply involved in melanoma invasion, metastasis, sensitivity to anticancer agents, and the like, and are useful as therapeutic targets. Therefore, not only the diagnosis of melanoma but also the application or application of the present invention to the treatment of melanoma is expected.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims

Melanoma-specific biomarker consisting of a biomolecule selected from the group consisting of PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene, CSS3 protein, BRP44L gene and BRP44L protein.
It is characterized in that the level in the sample of one or more biomolecules selected from the group consisting of PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene, CSS3 protein, BRP44L gene and BRP44L protein is used as an indicator. , Melanocyte tumor testing.
The melanocyte-based tumor examination method according to claim 2, comprising the following steps (1) to (3):
(1) a step of preparing a subject-derived specimen;
(2) detecting one or more of the biomolecules in the specimen; and (3) determining the malignancy of the melanocyte tumor based on the detection result.
For the PDZRN3 gene, PDZRN3 protein, DTX3L gene, DTX3L protein, CSS3 gene and CSS3 protein, according to the standard that the detection value is high and the malignancy is high, or that the possibility of onset is high if it can be detected,
The melanocyte according to claim 3, wherein the BRP44L gene and the BRP44L protein are determined in step (3) according to a criterion that the detection value is low and the malignancy is high, or a criterion that the detection is high and the malignancy is high. Tumor examination method.
The melanocyte-based tumor examination method according to claim 3 or 4, wherein the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value of the control sample.
The determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value in a sample collected in the past from the same subject. Melanocyte tumor testing.
The melanocyte-based tumor examination method according to any one of claims 2 to 6, wherein the specimen is skin tissue, blood, plasma, serum, urine, saliva, sweat, or tumor tissue.
A melanocyte-based tumor test reagent comprising a substance exhibiting specific binding to the melanoma-specific biomarker according to claim 1.
The melanocyte-based tumor test reagent according to claim 8, wherein the substance is an antibody.
A melanocyte tumor test kit comprising the melanocyte tumor test reagent according to claim 8 or 9.
A melanoma therapeutic agent comprising, as an active ingredient, a compound that suppresses the expression of a target gene selected from the group consisting of PDZRN3 gene, DTX3L gene and CSS3 gene.
The melanoma therapeutic agent according to claim 11, wherein the compound is a compound selected from the group consisting of the following (a) to (e):
(a) siRNA targeting the target gene;
(b) a nucleic acid construct that generates siRNA targeting the target gene in the cell;
(c) a single-stranded RNA having an expression suppressing sequence that suppresses expression of the target gene and a complementary sequence that anneals to the sequence;
(d) an antisense nucleic acid that targets the transcript of the target gene;
(e) Ribozymes that target transcripts of target genes.
Melanoma therapeutic agent containing the following (A) or (B) as an active ingredient and used in combination with an anticancer agent or a drug having an antitumor action:
(A) BRP44L protein or a part thereof (however, it has an effective effect on the anticancer drug sensitivity of melanoma cells);
(B) An expression vector carrying the BRP44L gene or a part thereof (however, it encodes a protein having an action effective for anticancer drug sensitivity of melanoma cells).
The melanoma therapeutic agent according to any one of claims 11 to 13, which is applied to a melanoma having resistance to a BRAF inhibitor.
A melanoma treatment method comprising the step of administering the melanoma therapeutic agent according to any one of claims 11 to 14 to a melanoma patient.