WO2008015942A1 - Therapeutic agent for neuroblastoma, method for screening for the therapeutic agent, and method for determination of prognosis of neuroblastoma - Google Patents

Abstract

A method for screening for a therapeutic agent for neuroblastoma, comprising the steps of: culturing a cell in the presence or absence of a compound to be tested; measuring the expression level of a Shf gene in a cultured cell; and determining the compound as being a therapeutic agent for neuroblastoma when the expression level of the Shf gene in a cell cultured in the presence of the compound is higher than that in a cell cultured in the absence of the compound.

Description

 Specification

 Neuroblastoma therapeutic agent and screening method thereof, and method for determining prognosis of neuroblastoma

 Technical field

 [0001] The present invention relates to a therapeutic agent for neuroblastoma, a screening method thereof, and a method for determining prognosis of neuroblastoma.

 Background art

 [0002] Neuroblastoma is a tumor that arises from the sympathetic nervous system (paraspinal sympathetic nerve trunk and adrenal medullary nerve cells), and is most common in malignant solid tumors in children. The most common sites of neuroblastoma are the adrenal gland, the retroperitoneum, the posterior mediastinum, the neck, the sympathetic ganglion of the pelvis, and the midline of the abdominal cavity. Neuroblastoma is extremely interesting clinically! / In cases where the onset age is less than 1 year of age, neuroblastoma has obvious spontaneous regression and maturation, while onset age is more than 1 year of age. In these cases, metastasis and growth of the tumor progress rapidly, resulting in a poor prognosis that is difficult to treat.

 [0003] In recent years, it has been clarified that there is a difference in gene expression level between a good prognosis subset and a poor prognosis subset of neuroblastoma. One of them is a gene encoding nerve growth factor receptor Trk A. The expression of TrkA gene is low in the poor prognosis group, whereas the expression is high in the good prognosis group (Non-patent Document 1).

 [0004] TrkA is present in the cell membrane, stimulated by nerve growth factor NGF, and transmits the stimulation as a signal via TrkA and various proteins in the cell. As a result, TrkA is thought to cause suppression of cell differentiation and proliferation. However, the molecular mechanism involved in TrkA in neuroblastoma remains unclear.

 [0005] In addition, the present inventors have developed novel genes from a plurality of neuroblastoma cDNA libraries in order to develop therapeutic methods for neuroblastoma, to identify genes involved in neuroblastoma development and biological characteristics thereof. About 5300 genes, including the above, have been isolated (Patent Documents 1 to 5 and Non-Patent Document 2). In addition, we have identified a number of genes that showed differential expression between the two subsets, the good prognosis group and the poor prognosis group.

[0006] Patent Document 1: Pamphlet of International Publication No. 01/66719 Patent Document 2: Pamphlet of International Publication No. 01/66733

 Patent Document 3: International Publication No. 02/97093 Pamphlet

 Patent Document 4: International Publication No. 02/103017 Pamphlet

 Patent Document 5: Pamphlet of International Publication No. 2004/39975

 Non-patent literature l: Nakagawara, Med. Pediatr. Oncol. 31, 1 13 (1998) Non-patent literature 2: OhiraM et al., Oncogene, 22, 5525-5536 (2003) Disclosure of the invention

 Problems to be solved by the invention

 [0007] Although neuroblastoma is the most frequent childhood malignant tumor, it is a disease with a mixed prognosis and good prognosis! The conventional treatment is a multidisciplinary treatment combining chemotherapy, radiation therapy, and surgical resection, but the prognosis is poor in advanced cases over 1 year of age, and there is no significant improvement in survival. If we can elucidate the molecular mechanisms that control neuroblastoma prognosis and develop therapeutics based on new mechanisms, we can broaden the range of treatment options for improving neuroblastoma prognosis. Accordingly, an object of the present invention is to provide a therapeutic agent for neuroblastoma and a screening method thereof.

 [0008] In addition, expression of TrkA in neuroblastoma is currently considered as one candidate for determining the prognosis of the disease. However, it is unclear how TrkA is involved in determining good and poor prognosis of neuroblastoma. Furthermore, even if the TrkA gene is highly expressed, the prognosis may not always be good if there is an abnormality in the TrkA intracellular signaling pathway. Therefore, an object of the present invention is to clarify the molecular mechanism that controls the prognosis of neuroblastoma and to provide a method for determining the prognosis of neuroblastoma.

 Means for solving the problem

[0009] Based on the results of an expression analysis in a clinical sample of neuroblastoma, the inventors of the present invention mediate the interaction between signal molecules, and a gene encoding Shf called adapter protein is highly expressed in a good prognosis group. Found that. Furthermore, the present inventors have found that the expression level of Shf gene is highly correlated with the prognosis of neuroblastoma, and in addition, is highly correlated with the expression level of TrkA gene. In addition, Shf and TrkA The inventors have shown that they co-localize and interact with each other. This suggests that Shf binds to TrkA to regulate TrkA signaling and may be involved in the determination of neuroblastoma prognosis. Based on the above findings, the present inventors have completed the present invention.

 [0010] That is, the present invention includes a step of culturing a cell under each condition in the presence and absence of a test compound, and a step of measuring the expression level of the Shf gene in each cultured cell. If the expression level of the Shf gene in the cells cultured in the presence of the test compound is higher than the expression level of the Shf gene in the cells cultured in the absence of the test compound, the test compound is And a method for screening a therapeutic agent for neuroblastoma, comprising the step of determining that the therapeutic agent is a neuroblastoma. The screening method of the present invention is based on the knowledge newly discovered by the present inventor that the expression level of Shf gene strongly correlates with good prognosis and poor prognosis of neuroblastoma. This makes it possible to develop therapeutic agents for improving the prognosis of neuroblastoma.

 [0011] The screening method of the present invention also includes a step of culturing cells in the presence and absence of a test compound, and the Shf gene and TrkA gene in each cultured cell. The expression level of Shf gene and TrkA gene in cells cultured in the presence of the test compound !, Shf in cells cultured in the absence of the test compound And a step of determining the test compound as a therapeutic agent for neuroblastoma when the expression level of the gene and the TrkA gene is higher. With the above configuration, a therapeutic drug for improving the prognosis of neuroblastoma based on the finding that the expression level of Shf gene is highly correlated with the prognosis of neuroblastoma and also highly correlated with the expression level of TrkA gene Can be developed.

[0012] The screening method of the present invention also comprises a step of culturing cells in the presence and absence of a test compound, and the mutual relationship between Shf and TrkA in each cultured cell. In the presence of the test compound! /, The interaction force of Shf and TrkA in the cells cultured in the presence of the test compound S, Shf and in the cells cultured in the absence of the test compound And a step of determining the test compound as a therapeutic agent for neuroblastoma when the interaction is stronger than TrkA interaction. The screening method of the present invention It applies the molecular mechanism that Shf and TrkA interact with each other. This molecular mechanism has been newly discovered by the present inventor, which enables the control of Trk A signaling by Shf and the new molecular mechanism based on the new molecular mechanism. Development is possible.

 [0013] It is preferable that the cell to be used in the screening method of the present invention is a cell derived from a clinical sample of human neuroblastoma. Preferably, the cell is derived from a clinical sample of human neuroblastoma having a poor prognosis. It is preferable. This makes it possible to develop therapeutic agents for improving the prognosis of human neuroblastoma, particularly for the treatment of patients with poor prognosis.

 [0014] The present invention provides a therapeutic agent for neuroblastoma comprising a vector having a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 1. The present invention also relates to the use of a vector having a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 1 for the manufacture of a therapeutic agent for neuroblastoma, and to a neuroblastoma patient described in SEQ ID NO: 1. Provided is a method for treating neuroblastoma, comprising a step of administering a vector having a nucleic acid comprising a base sequence. Furthermore, the present invention provides a therapeutic agent for neuroblastoma comprising a vector having a nucleic acid encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. The present invention also relates to the use of a vector having a nucleic acid encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 for the manufacture of a therapeutic agent for neuroblastoma, and to a neuroblastoma patient, SEQ ID NO: 2 A method for treating neuroblastoma comprising the step of administering a vector having a nucleic acid encoding a protein comprising the amino acid sequence described in 1. Here, the base sequence described in SEQ ID NO: 1 corresponds to the Shf gene, and the amino acid sequence described in SEQ ID NO: 2 corresponds to Shf. The Shf gene and Shf GenBank Accession No. is NM138356. The prognosis improvement of neuroblastoma disease can be expected by the expression of Shf gene and the enhancement of Shf function.

[0015] The therapeutic agent of the present invention further includes a vector having a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 3 or a vector having a nucleic acid encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. Is preferred. Here, the base sequence described in SEQ ID NO: 3 corresponds to the Trk A gene, and the amino acid sequence described in SEQ ID NO: 4 corresponds to TrkA (GenBank Accession No .: NP002529). Improves the prognosis of neuroblastoma disease by enhancing the function of Shf and TrkA and normal functioning of TrkA signaling involving Shf Can be expected.

 [0016] The present invention further comprises a step of measuring the expression level of Shf gene in a clinical sample of human neuroblastoma with unknown prognosis, and the expression level of Shf gene with a good prognosis and a poor prognosis. A method for determining the prognosis of human neuroblastoma, comprising the step of comparing the expression level of the Shf gene in a clinical sample of neuroblastoma. The determination method of the present invention is based on the finding that the expression level of the Shf gene is strongly correlated with the survival rate of patients with neuroblastoma, that is, with a good prognosis and poor prognosis of neuroblastoma. Such a molecular mechanism has been newly discovered by the present inventor, and this makes it possible to determine the prognosis of human neuroblastoma whose prognosis is unknown.

 [0017] The determination method of the present invention comprises a step of measuring the expression level of Trk A gene in a clinical sample of human neuroblastoma of unknown prognosis, and the expression level of TrkA gene is determined with good prognosis and poor prognosis. It is preferable to further comprise a step of comparing the expression level of the TrkA gene in a clinical sample of human neuroblastoma. The determination method of the present invention is an application of the knowledge of the present inventors that the expression level of Shf gene is highly correlated with the prognosis of neuroblastoma, and also highly correlated with the expression level of TrkA gene. It is. By analyzing the expression level of the TrkA gene in addition to the analysis of the Shf gene, the prognosis of human neuroblastoma with an unknown prognosis can be determined with higher reliability.

 The invention's effect

 [0018] According to the screening method of the present invention, it is possible to develop a therapeutic agent for improving the prognosis of neuroblastoma, which has a different mechanism of action from the past. In addition, the therapeutic agent of the present invention can be expected to improve the prognosis of neuroblastoma disease. Furthermore, the determination method of the present invention makes it possible to determine the prognosis of a human neuroblastoma whose prognosis is unknown. By combining the determination method of the present invention with the screening method and / or therapeutic agent, it becomes possible to develop a therapeutic agent and select a therapeutic method in accordance with the prognosis good and poor prognosis of neuroblastoma. Brief Description of Drawings

[0019] [Fig. 1] Compared to electrophoretic photographs showing the measurement results of the expression level of Shf mRNA in clinical samples of neuroblastoma (eight samples each with good prognosis and poor prognosis) by semi-quantitative RT-PCR. FIG.

 FIG. 2 is a graph showing the correlation between Shf mRNA expression level and prognosis.

 FIG. 3 is a graph showing the correlation between expression levels of TrkA mRNA and Shf mRNA.

 FIG. 4 is a diagram corresponding to an electrophoretic photograph showing the measurement result of the expression level of Shf mRNA in a normal tissue by a semiquantitative RT-PCR method.

FIG. 5 is a diagram corresponding to a stained image showing expression of Shf mRNA by an in situ hybridization method. sp, spinal cord; DRG, dorsal root ganglion; dien _{C e} phalon, diencephalon.

 FIG. 6 is a diagram corresponding to a stained image showing the localization of Shf and TrkA in rat pheochromocytoma PC 12 cells by immunostaining experiments.

 FIG. 7 is a view corresponding to a Western plot image showing the interaction between Shf and TrkA by a coimmunoprecipitation experiment. Shf and TrkA expression constructs were cotransfected into H1299 cells. Anti-Shf antibody and anti-TrkA antibody were used for immunoprecipitation and Western blot analysis.

 FIG. 8 is a schematic diagram showing a signal transduction mechanism model of Shf and TrkA.

 BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, preferred embodiments of the present invention will be described in detail.

 [0021] (Method for screening therapeutic agents)

 The therapeutic drug screening method in the present embodiment includes a screening method for the first and second therapeutic drugs using the expression level of the Shf gene as an index, and a third treatment using the interaction between Shf and TrkA as an index. There are drug screening methods.

 [0022] First, a method for screening a therapeutic agent using the expression level of the Shf gene as an index will be described.

 The first therapeutic drug screening method involves the step of culturing cells in the presence and absence of a test compound, and the expression level of Shf gene in each cultured cell. When the measurement step and the expression level S of Shf gene in cells cultured in the presence of the test compound are higher than the expression level of Shf gene in cells cultured in the absence of the test compound, Determining the test compound as a therapeutic agent for neuroblastoma.

[0023] Here, the expression level of the gene means the expression level of mRNA that is a transcription product of the gene and / or Indicates the expression level of the protein which is the translation product. The measurement of mRNA expression can be performed using a measurement system known to those skilled in the art. Specifically, quantitative RT-PCR, quantitative real-time RT-PCR, quantitative northern blot And the like, and quantitative ribonuclease protection method. The protein expression level may be measured using a measurement system known to those skilled in the art, for example, quantitative Western blotting and ELISA. As a control, the expression level of the target gene such as the Shf gene is standardized using the expression level of mRNA and / or protein such as GADPH, which is a housekeeping gene, and beta-actin. Alternatively, the expression level of the target gene and / or control gene in a plurality of samples collected from the same target force and / or the same sample may be measured, and the expression level may be obtained from the average value of each. By using these methods, it is possible to quantitatively measure gene expression.

 [0024] A test compound in which the expression level of the Shf gene in the presence of the test compound is larger than the expression level of the Shf gene in the absence of the test compound can be determined as a therapeutic agent for neuroblastoma. The therapeutic agent is most preferably an agent that improves the prognosis of neuroblastoma, but any agent that improves the prognosis is suitable for the purpose of the present invention.

 [0025] In the second therapeutic drug screening method, it is preferable to analyze the expression level of the TrkA gene, which is a candidate gene involved in the prognosis determination of neuroblastoma disease, in addition to the expression level of the Shf gene. In yet another embodiment, expression levels of genes that are differently expressed between two subsets of a well-known group and a poor-prognosis group, which are conventionally known, may be analyzed simultaneously.

 [0026] The cells used for screening may be cultured cells such as H1299 derived from human lung cancer cells, PC 12 derived from rat adrenal pheochromocytoma, or may be cultured cells derived from neural tissue. Further, it is more preferable that the cultured cell derived from neuroblastoma is a cell derived from a preferred clinical sample of human neuroblastoma, more preferably a cell derived from a clinical neuron of human neuroblastoma having a poor prognosis. .

[0027] Test compounds include, but are not limited to, low molecular weight compounds, peptides, proteins, nucleic acids (DNA, RNA, PNA) and the like. Also, for screening, any screen Compound library may be used. It should be noted that cell culture conditions and test compound administration conditions can be appropriately adjusted by those skilled in the art.

 [0028] Next, a third therapeutic drug screening method using the interaction between Shf and TrkA as an index will be described. A third method for screening for therapeutic agents is a step of culturing cells in each condition in the presence and absence of a test compound, and a step of measuring the interaction of Shf and TrkA in each cultured cell. And the interaction force S of Shf and TrkA in the cells cultivated in the presence of the test compound, and the interaction force Shf and TrkA in the cells cultivated in the absence of the test compound, And determining the test compound as a therapeutic agent for neuroblastoma.

 [0029] Shf and TrkA expressed in cells are preferably endogenous, but in this case, Shf and TrkA can be expressed by tags such as GST and HA. May be labeled with a fluorescent protein which may be fused. Interaction refers to direct and / or indirect interaction between proteins, where Shf and Tr kA form a complex or are in close proximity to be functionally linked. It means doing. For the measurement of the interaction, a system for measuring protein interactions known to those skilled in the art can be used. Specifically, the measurement by co-immunoprecipitation method or fluorescence resonance energy transfer (FRET) is applied. Measurement and the like. The obtained quantitative measurement value is compared between the presence and absence of the test compound to determine the utility of the test compound as a therapeutic agent for neuroblastoma.

 [0030] (Therapeutic)

 Further, the therapeutic agent for neuroblastoma, which is an embodiment of the present invention, includes a vector having the Shf gene (SEQ ID NO: 1). In yet another embodiment, the therapeutic agent comprises a vector having a nucleic acid encoding Shf (SEQ ID NO: 2). In a further embodiment, the therapeutic agent preferably further comprises a vector having a TrkA gene (SEQ ID NO: 3) or a vector having a nucleic acid encoding TrkA (SEQ ID NO: 4).

[0031] Vectors can be prepared based on DNA or RNA viruses. Any virus vector such as a MoMLV vector, a herpes virus vector, an adenovirus vector, an AAV vector, an HIV vector, an SIV vector, or a Sendai virus vector may be used. In addition, one or more of the viral protein component proteins are replaced with a heterologous virus component protein, or a part of the base sequence constituting the genetic information is replaced with a heterologous virus base sequence. A pseudo-type virus vector can also be used in the present invention. For example, a pseudo-type virus vector in which the Env protein, which is the coat protein of HIV, is replaced with the VSV-G protein, which is the coat protein of the small vesicular stomatitis virus (VSV). Furthermore, viruses having a host range other than humans can be used as virus vectors as long as they have a therapeutic effect. As a vector other than a virus, a complex of calcium phosphate and nucleic acid, a liposome, a cationic lipid complex, a Sendai virus ribosome, a polymer carrier having a polycation as a main chain, and the like can be used.

 [0032] Further, the expression cassette used for the expression of the gene in the vector can be used without particular limitation as long as it can express the gene in the target cell. One skilled in the art can readily select such an expression cassette. Preferably, it is an expression cassette capable of gene expression in animal-derived cells, more preferably an expression cassette capable of gene expression in mammal-derived cells, and particularly preferably in human-derived cells. This is an expression cassette capable of gene expression. The gene promoter used in the expression cassette is, for example, adenovirus, cytomegalovirus, human immunodeficiency virus, simian virus 40, rous sarcoma virus, herpes simplex virus, mouse leukemia virus, symbis virus, hepatitis A Virus, Hepatitis B virus, Hepatitis C virus, Papilloma virus, Human T-cell leukemia virus, Influenza virus, Japanese encephalitis virus, JC virus, Parvovirus B19, Poliovirus-derived promoter, albumin, SR a, promoters derived from mammals such as heat shock proteins and elongation factors, chimeric promoters such as CAG promoters, promoters whose expression is induced by tetracycline, steroids, and the like.

 [0033] (Method for determining prognosis of neuroblastoma)

The method of determining the prognosis of neuroblastoma in the present embodiment includes a step of measuring the expression level of Shf gene in a clinical sample of human neuroblastoma with an unknown prognosis, and the expression level of Shf gene with a good prognosis, and Shf remains in clinical samples of human neuroblastoma with poor prognosis And a step of comparing with the expression level of the gene.

 [0034] The prognosis is determined based on the knowledge of the present invention that the prognosis of neuroblastoma is good when the expression level of the Shf gene is high. The expression level of Shf gene in clinical samples of human neuroblastoma with good prognosis is a statistically processed measurement of the expression level obtained from multiple cases with good prognosis (good prognosis group). However, the same is true for the expression level of the Shf gene in clinical samples of human neuroblastoma with poor prognosis. Regarding the determination of good prognosis, specifically, the expression level of Sh f gene in clinical samples of human neuroblastoma with unknown prognosis is the same as the expression of Shf gene in clinical samples of human neuroblastoma with good prognosis. If the amount is higher than the dose and / or statistically falls within the distribution range of the good prognosis group in comparison with the expression level obtained from the good prognosis group, the prognosis of the subject is determined to be good. can do. In addition, judgment of poor prognosis can be performed in the same way as judgment of good prognosis.

 [0035] As used herein, "good prognosis" refers to a state of a neuroblastoma where the tumor is localized or has become a regression or benign sympathetic ganglion cell, N — Judging from myc and other tumor markers, the grade is low. The N-myc gene is usually only one per haploid in normal cells and neuroblastomas with good prognosis, whereas in neuroblastoma with poor prognosis, it is amplified several tens of times. Is an oncogene. In a preferred embodiment of the present invention, stage 1, 2 or 4 s based on the international neuroblastoma staging, age of onset is less than age, and survives 5 years or more after surgery without recurrence. However, this is not limited to such a specific example, in which N-myc amplification is not recognized as a good prognosis in human neuroblastoma.

 [0036] The term "poor prognosis" as used herein refers to a state in which tumor progression is observed among neuroblastomas, and is highly malignant as judged from N-myc and other tumor markers. It is judged. In a preferred embodiment of the present invention, humans are those whose stage 3 or 4 has an onset age of 1 year or more, died within 3 years after surgery, and N-myc amplification was observed in clinical tissues. Force S, which is a poor prognosis in neuroblastoma, is not limited to such a specific example.

[0037] In another embodiment, the method for determining the prognosis of neuroblastoma includes the expression level of the Shf gene, Ability to analyze the expression level of the TrkA gene, which is a candidate gene for determining the prognosis of neuroblastoma disease. In yet another embodiment, the expression levels of genes that are differently expressed between two subsets of a well-known group and a poor-prognosis group, which are conventionally known, may be analyzed simultaneously.

 Example

 [0038] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

 [0039] (Experimental material)

 Superscript II reverse transcriptase was purchased from LifeTechnologies. Random primers were purchased from Takara Sake Brewery. TaqMan (registered trademark) Universal PCR Master Mix was purchased from Perkin—Elmer Applied Biosystems. Shf probed primers were purchased from Applied Biosystems. Anti-Shf antibody was purchased from MBL. Anti-Trk antibody was purchased from SANTA CRUZ BIOTECHNOLOGLY, and anti-Tubul in antibody was purchased from Neo MARKERS.

 [0040] The various primers used in the RT-PCR method and the Real-time RT-PCR method are as follows.

 Shf: 5 '-TATGAGCCAGAGAGGAGGATGG-3' (SEQ ID NO: 5)

 5 '-CTGTCCAGCTGTCCCACAGGTG-3' (SEQ ID NO: 6) GAPDH: 5 '-ACCTGACCTGCCGTCTAGAA-3' (SEQ ID NO: 7)

 5 '-TCCACCACCCTGTTGCTGTA-3' (SEQ ID NO: 8)

[0041] (Experimental method)

 The following method was performed according to each document.

 The semi-quantitative RT-PCR method, immunostaining method and immunoprecipitation method are the same as those described in Hanamoto et al., J. Biol. Chem., 280: 16665-166675, 2005. The experimental system was optimized. Quantitative real—time RT—PCR method and in situ nof, redisaction method (Machida et al., Oncogene., 25: 1931— 1942, 2006. The experimental system was optimized.

[0042] (Example 1: Expression analysis by RT-PCR method) Total RNA was prepared from clinical samples of human neuroblastoma with good prognosis and poor prognosis, and the expression level of Shf mRNA was analyzed by semi-quantitative RT-PCR using these total RNAs as templates. The results are shown in Fig. 1. It was confirmed that the Shf gene was highly expressed in the good prognosis group, suggesting that there is a functional relationship between the expression level of the Shf gene and the prognosis of neuroblastoma.

[0043] (Example 2: Survival analysis by force plan Meyer method)

 Quantitative real-time RT PCR was performed on 105 clinical samples collected from patients with neuroblastoma to examine the relationship between Shf mRNA expression levels and patient survival after diagnosis. Figure 2 shows the results of analysis by the force plan Meyer method. There was a significant correlation (p = 0.045) between the good prognosis group and the high expression of Shf. This proved that the prognosis was good in the case of high expression of Shf mRNA.

[Example 3: Correlation of Shf mRNA and TrkA mRNA expression]

 Furthermore, quantitative real-time RT-PCR was performed using clinical samples of neuroblastoma, and the expression level of Shf mRNA was compared with the expression level of TrkA mRNA. As a result, as shown in Fig. 3, both were significantly correlated (p <0.00055), and in cases where the mRNA of the candidate gene TrkA involved in prognosis was highly expressed, Shf mRNA was significantly high expressed. It was shown that

(Example 4: Confirmation of tissue specificity of Shf mRNA expression)

 Figure 4 shows the results of comparing the expression level of Shf mRNA in each normal tissue by semi-quantitative RT-PCR. High expression of Shf mRNA was observed in the brain, cerebellum and fetal brain. Furthermore, Fig. 5 shows the results of analysis by the in situ hybridization method. Embryonic 13. Examination of Shf mRNA expression tissue on day 5 revealed expression in the spinal cord, dorsal root ganglia and diencephalon. Therefore, it was suggested that the Shf gene is expressed in the nervous system and is involved in the maintenance and differentiation of neural tissues.

[Example 5: Confirmation of intracellular localization of Shf and TrkA by immunostaining experiment]

In order to clarify the intracellular localization of Shf and TrkA, immunostaining experiments using rat pheochromocytoma PC12 cells revealed that endogenous Shf and TrkA colocalized in the cytoplasm. became. Fig. 6 shows the results of immunostaining analysis. TrkA is transmembrane type Considering that it is a receptor for Sf, it was suggested that Sh f and TrkA co-localize in the cytoplasm and / or cell membrane and interact functionally with each other.

 (Example 6: Detection of Shf and TrkA immune complex formation)

 H1299 cells were cotransduced with Shf and TrkA expression constructs. After the cultivation, the cell suspension was fractionated into a soluble fraction and an insoluble fraction, and the obtained soluble fraction was subjected to an immunoprecipitation experiment. Both Shf and TrkA were detected in the soluble fraction. Figure 7 shows the analysis results of the coimmunoprecipitation experiment. From this result, it was shown that Shf and TrkA co-expressed together in H1299 cells form an immune complex.

 [0048] FIG. 8 is a schematic diagram showing a signal transduction mechanism model of Shf and TrkA. In nerve cells, when nerve growth factor NGF binds to cell membrane-like nerve growth factor receptor TrkA, the signal is transmitted to the inside of the cell and to the nucleus, and responses such as nerve cell differentiation and cell growth arrest occur. Considering the findings obtained by the present invention, Shf is involved in the NGF / TrkA signal by binding to TrkA and is further involved in determining the prognosis of neuroblastoma by transmitting the signal downstream. The possibility of! / Was suggested. These findings suggest that research and development targeting Shf may be useful in the development of new therapeutic agents for neuroblastoma with poor prognosis and other nervous system tumors, as well as clinical treatment. It is expected to improve the choice of law and apply to custom-made medicine.

Claims

The scope of the claims  [1] A step of culturing cells under each condition in the presence and absence of a test compound!  Measuring the expression level of the Shf gene in each cultured cell;  When the expression level of Shf gene in cells cultured in the presence of the test compound is higher than the expression level of Shf gene in cells cultured in the absence of the test compound, the test compound is A step of determining a therapeutic agent for neuroblastoma;  A screening method for a therapeutic agent for neuroblastoma.  [2] a step of culturing the cells under the respective conditions in the presence and absence of the test compound! /  Measuring the expression level of Shf gene and TrkA gene in each cultured cell;  Expression level of Shf gene and TrkA gene in cells cultured in the presence of the test compound When the expression level of Shf gene and TrkA gene is higher in the cells cultured in the absence of the test compound, Determining the test compound as a therapeutic agent for neuroblastoma;  A screening method for a therapeutic agent for neuroblastoma. [3] a step of culturing cells under each condition in the presence and absence of the test compound! /  The step of measuring the interaction of Shf and TrkA in each cultured cell, and the interaction force of Shf and TrkA in the cell cultured in the presence of the test compound In the cell cultured in the absence of the test compound Determining that the test compound is a therapeutic agent for neuroblastoma when the interaction of Shf and TrkA is stronger than  A screening method for a therapeutic agent for neuroblastoma.  [4] The screening method according to any one of [1] to [3], wherein the cells are cells derived from a clinical sample of human neuroblastoma. [5] A therapeutic agent for neuroblastoma, comprising a vector having a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1. [6] A therapeutic agent for neuroblastoma, comprising a betater having a nucleic acid encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. [7] The therapeutic agent according to claim 5 or 6, wherein the therapeutic agent further comprises a vector having a nucleic acid consisting of the base sequence set forth in SEQ ID NO: 3. [8] The therapeutic agent according to claim 5 or 6, wherein the therapeutic agent further comprises a vector having a nucleic acid encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4. [9] measuring the expression level of Shf gene in clinical samples of human neuroblastoma with unknown prognosis;  Comparing the expression level of the Shf gene with the expression level of the Shf gene in a clinical sample of human neuroblastoma with good and poor prognosis;  A method for determining the prognosis of human neuroblastoma. [10] measuring the expression level of the TrkA gene in a clinical sample of human neuroblastoma of unknown prognosis;  Comparing the expression level of the TrkA gene with the expression level of the TrkA gene in human neuroblastoma clinical samples with good and poor prognosis;  The prognosis determination method for human neuroblastoma according to claim 9, further comprising: