JP6162958B2 - Determination of malignancy and prognosis of neuroblastoma by CDX1 measurement - Google Patents

Description

  The present invention relates to determination of malignancy of neuroblastoma by CDX1 and prognosis determination. The present invention also relates to a method for detecting cells capable of forming neuroblastoma spheres in a subject. Furthermore, the present invention relates to a method for screening a compound that inhibits the activity of CDX1.

  In the field of cancer treatment, cancer stem cells have recently attracted attention. A cancer stem cell is a cancer cell having the properties of a stem cell, is drug resistant, and has a high ability to regenerate cancer. Even if most cancer cells are killed by chemotherapy or the like, if cancer stem cells having drug resistance are present, cancer can recur from there. Therefore, if cancer stem cells can be specifically treated, recurrence of cancer can be effectively prevented and useful for treatment.

  Several reports have been made as markers for cancer stem cells, and among them, it has been reported that CD133 is expressed in cancer stem cells (Non-patent Documents 1-8). Therefore, CD133 is considered to be a marker for cancer stem cells. However, there are also reports of CD133 negative cancer stem cells in colon cancer and glioma (Non-patent Documents 9-12). CD133 is one of cancer stem cell markers for colon cancer and brain tumor, but other cancer stem cell markers (CD44). , ALDH, sphere formation).

  CD133 is a 5-transmembrane protein that has been identified as AC133 antigen and has been confirmed to be expressed in CD34-positive blood stem cells (Non-patent Document 13). The chromosomal site of CD133 (also referred to as Prominin 1) is human 4p15.33, mouse chromosome 5. Human CD133 has 865 amino acids and a molecular weight of 120 kDa. Prominin 1 is expressed in transformable intestinal stem cells (Non-patent Document 14).

CD133 has been reported to affect the maintenance of the retina, and loss of CD133 causes abnormal disk morphogenesis and photoreceptor degeneration (Non-patent Document 15). In addition, a frameshift mutation in human CD133 causes human retinal degeneration (Non-patent Document 16). In addition, CD133 gene mutations were found in patients with genetic diseases of retinal degeneration.
However, these reports do not elucidate the function of CD133 in carcinogenesis or cancer refractory.

  There are several reports on the transcriptional regulation of CD133. The 5′-UTR structure of the CD133 gene is as shown in FIG. 1, and it has been reported that CD133 expression is driven by different promoters and is mainly controlled by DNA methylation (Non-patent Documents 17-21). Further, it has been reported that CD133 has promoters P1 to P5 (Non-patent Document 17).

  CD133 expression has been reported in stem cell-like neuroblastoma (NB) cells. Walton et al. Suggested that CD133 may have a role in neuroblastoma stem cells (Non-patent Document 22).

  Neuroblastoma is a type of pediatric cancer in which neuroblasts proliferate abnormally during growth. Neuroblastoma is the most common refractory tumor among extracranial solid tumors, and is the malignant tumor that has the highest number of cases following childhood solid tumors after brain tumors. The annual number of cases in Japan is estimated to be around 200 to 250. The prognosis of early cases is generally good, but the prognosis of advanced cases is still poor at about 30% in INSS (International Neuroblastoma Staging System) stage IV in Japan. There are clinically and biologically distinct subsets of neuroblastoma. Neuroblastomas with good prognosis are generally less than 12 months old and have low recurrence. Neuroblastoma with a poor prognosis has many cases after 12 months of age and progresses quickly. Therefore, if it can be known at an early stage whether the prognosis of neuroblastoma is good or bad, it can be useful for treatment.

  Hansford et al. Reported that neuroblastoma contains tumor progenitor cells (Non-patent Document 23). According to Hansford et al., Cells released from the tumor grew as spheres under certain conditions (Id.). This neuroblastoma sphere has a high tumorigenic potential, and it is described that even 10 passage tumor sphere cells derived from a highly malignant neuroblastoma can form a neuroblastoma when injected into mice. It has also been reported that CD133 is up-regulated in neuroblastoma spheres (Non-patent Document 24).

  Then, although the cancer stem cell marker CD133 was interested in the function in neuroblastoma, the present inventors confirmed that CD133 suppresses neuroblastoma cell differentiation by modifying the signal transduction pathway (Non-patent Document 25). ). CD133 is expressed in multiple neuroblastoma cells. Moreover, the survival rate is different between the group with high CD133 expression level and the group with low CD133 expression level in children over 1 year old, and the prognosis is poor in the group with high CD133 expression level. CD133 also upregulates neuroblastoma cell proliferation. Conversely, when CD133 is knocked down, neuroblastoma cell proliferation and tumor growth are suppressed, and colonies do not form on the soft agar medium. Furthermore, neuroblastomas knocked down with CD133 do not form tumors when transplanted into mice. Moreover, CD133-related neuroblastoma cell differentiation and RET suppression depend on the p38MAPK and PI3K / AKT pathways (Non-patent Document 25). Non-Patent Document 25 further describes that CD133 suppresses RET transcription in neuroblastoma. RET is a GDNF receptor involved in the development and differentiation of the sympathetic adrenal system (Non-patent Document 26).

  To understand the oncogenic mechanism of neuroblastoma and the nature of cancer stem cells, it is necessary to elucidate the CD133 expression control mechanism specific to the neuroblastoma sphere. In addition, it is necessary to elucidate the CD133 signal pathway.

Singh et al., Nature. 2004 Nov 18; 432 (7015): 396-401. Beier et al., Cancer Res. 2007 May 1; 67 (9): 4010-5. O’Brien et al., Nature. 2007 Jan 4; 445 (7123): 106-10. Ricci-Vitiani et al., Nature. 2007 Jan 4; 445 (7123): 111-5. Collins et al., Cancer Res. 2005 Dec 1; 65 (23): 10946-51. Ito et al., Nature. 2008 Jun 19; 453 (7198): 1072-8. Ma S et al., Gastroenterology. 2007 Jun; 132 (7): 2542-56. Ma S et al., Oncogene. 2008 Mar 13; 27 (12): 1749-58. Shmelkov SV et al. J Clin. Invest. 118, 2021-4 (2008) Dalerba P et al. Proc. Nat, Acad. Sci. USA 104: 10158-63 (2007) Chu P et al. Int. J. Cancer 124: 1312-21 (2009) KELLY JJP et al., STEM CELLS 2009; 27: 1722-1733 Yin AH et al., Blood. 1997 Dec 15; 90 (12): 5002-12. Zhu L et al. Nature 457, 603-7 (2009) S. Zacchigna et al. J. Neuroscience, 2009, 29, 2297-2308 Maw M et al. Human Molecular genetics, 2000, 9, 27-34. Shmelkov SV et al., Blood. 2004 Mar 15; 103 (6): 2055-61. Tabu K et al., Cell Res. 2008 Oct; 18 (10): 1037-46. Baba T et al., Oncogene. 2009 Jan 15; 28 (2): 209-18. Pleshkan VV et al., Biochim Biophys Acta. 2008 Oct; 1779 (10): 599-605. Yi JM et al., Cancer Res. 2008 Oct 1; 68 (19): 8094-103. Walton et al., Neoplasia. 2004 Nov-Dec; 6 (6): 838-45. Hansford et al., Cancer Res. 2007 Dec 1; 67 (23): 11234-43. Coulon A et al., Neoplasia. 2011 October; 13 (10): 991-1004. Takenobu H et al., Oncogene. 2011 Jan 6; 30 (1): 97-105 Klein, 1994, FASEB J. 1994 Jul; 8 (10): 738-44.

  An object of the present invention is to provide a method for determining the malignancy of a neuroblastoma or a method for determining the prognosis of a neuroblastoma. Another object of the present invention is to provide a method for detecting cells capable of forming neuroblastoma spheres. Furthermore, an object of the present invention is to provide a method for screening a drug for neuroblastoma.

The present inventors focused on CD133 whose expression level is increased in the neuroblastoma sphere, and as a result of earnest research on elucidation of the CD133 expression control mechanism specific to the neuroblastoma sphere, CDX1 was expressed as CD133. The present invention has been completed. Furthermore, the present inventors also found that CDX1 regulates the expression of OCT4, and completed the present invention.
That is, the present invention is as follows.

[1] A method for assisting in determining the malignancy of neuroblastoma and / or a method for assisting in determining the prognosis of neuroblastoma,
(A) determining the expression level of the CDX1 gene in a sample obtained from the subject;
(B) comparing the determined CDX1 gene expression level with a standard;
Wherein, if the expression level of the CDX1 gene is higher than the standard, it is determined that the neuroblastoma has a high malignancy or is determined to have a poor prognosis.
[2] A method for detecting cells capable of forming neuroblastoma spheres in a subject comprising the steps of:
(A) determining the expression level of the CDX1 gene in a sample obtained from the subject;
(B) comparing the determined CDX1 gene expression level with a standard;
Wherein the expression level of the CDX1 gene in the sample is higher than the standard indicates the presence of cells capable of forming neuroblastoma spheres in the subject.
[3] To determine neuroblastoma malignancy, including means to determine CDX1 gene expression levels, to determine neuroblastoma prognosis, or to detect cells that can form neuroblastoma spheres Kit.
[4] The means for determining the expression level of the CDX1 gene is a primer for detecting the expression of the CDX1 gene, a labeled nucleic acid that targets the mRNA of the CDX1 gene, or a labeled anti-CDX1 antibody. ] Kit described in.
[5] A method for screening a compound that inhibits CDX1, comprising the following steps:
(A) providing a CDX1 gene expression product or a nucleic acid construct having a CDX1 gene and a candidate compound to a cell capable of forming a neuroblastoma sphere;
(B) the cells are cultured under conditions capable of forming a neuroblastoma sphere, and when the nucleic acid construct having a CDX1 gene in (a) is used, the CDX1 gene in the nucleic acid construct can be expressed. ,
(C) counting the number of neuroblastoma spheres formed, and (d) comparing the number measured in (c) above with a positive control,
A positive control is a cell that can form neuroblastoma spheres under conditions that can form neuroblastoma spheres in the absence of a candidate compound and in the presence of a CDX1 gene expression product or a nucleic acid construct having a CDX1 gene and (a ) Is the number of neuroblastoma spheres formed when cultured under conditions where the CDX1 gene in the nucleic acid construct can be expressed,
Said method, wherein the formation of neuroblastoma spheres is suppressed compared to a positive control, indicating that the candidate compound inhibits CDX1.
[6] A screening method for a compound that inhibits CD133 transcription-promoting activity of CDX1, comprising the following steps:
(A) adding an expression product of the CDX1 gene and a candidate compound to a transformed cell or model non-human animal containing a nucleic acid construct having the promoter P1 of CD133 operably linked to a reporter gene;
(B) culturing or growing the cell or model non-human animal under conditions where the reporter gene can be expressed; and (c) detecting the expression of the reporter gene.
Wherein the reporter gene expression is not detected indicates that the candidate compound inhibits CD133 transcription-promoting activity of CDX1.
[7] The method according to [6], wherein the promoter P1 of CD133 has the base sequence described in SEQ ID NO: 30 or SEQ ID NO: 38.
[8] A screening method for a compound that inhibits the OCT4 transcription promoting activity of CDX1, comprising the following steps:
(A) adding a CDX1 gene expression product and a candidate compound to a transformed cell or model non-human animal comprising a nucleic acid construct having an OCT4 promoter sequence operably linked to a reporter gene;
(B) culturing or growing the cell or model non-human animal under conditions where the reporter gene can be expressed; and (c) detecting the expression of the reporter gene.
Wherein the expression of the reporter gene is not detected indicates that the candidate compound inhibits the OCT4 transcription promoting activity of CDX1.
[9] The promoter sequence of OCT4 has the sequence of promoter A and / or D of OCT4, the promoter A of OCT4 has the base sequence shown in SEQ ID NO: 32, and / or the promoter D of OCT4 has the sequence The method according to [8], having the base sequence described in No. 33.
[10] The method according to any one of [5] to [9], wherein the CDX1 gene is a human CDX1 gene having the nucleotide sequence set forth in SEQ ID NO: 34.

  Measuring the expression level of the CDX1 gene can determine the grade of neuroblastoma, determine the prognosis of neuroblastoma, or detect cells that can form neuroblastoma spheres in a subject can do.

  A compound that inhibits the activity of CDX1 can be screened by utilizing the CD133 or OCT4 transcription promoting activity of CDX1. Thus, an inhibitor of CDX1 can be identified and used for the treatment of neuroblastoma.

It is a figure which shows 5'-UTR structure of CD133 gene. The positional relationship between the CD133 gene and plasmids pGL4.17-2.5k, pGL4.17-1.8k, pGL4.17-1.1k, pGL4.17-0.6k is shown. The positional relationship between the CD133 gene and plasmids pGL4.17-1.1k, pGL4.17-0.4k, and pGL4.17-0.7k is shown. The result of searching for a sphere-specific CD133 promoter is shown. The result of the promoter assay of CD133 is shown. The results of a more detailed CD133 promoter assay are shown. CD133 activates the CD133 P1 promoter. The transcription factor binding site present in P1 (1.1k) of CD133 is shown. Many CDX1 binding sites were observed. The results of examining transcription factors whose expression is specifically increased in the sphere are shown in the upper part. It was confirmed that the expression of transcription factor CDX1 was increased. The bottom row shows the quantitative results that CDX1 gene is expressed in a sphere-specific manner. The result of having confirmed the expression induction of CD133 by CDX1 using quantitative PCR is shown. The result of having confirmed the expression induction of CD133 by CDX1 by the Western blot is shown. The results of examining the transcription of CD133 ORF, CD133 exon 1A, and CD133 exon 1B when CDX1 was introduced are shown. The results of examining the transcription of CD133 when CDX1 was knocked down are shown. The result of ChIP assay is shown. It was confirmed that CDX1 acts by directly binding to the promoter P1 of CD133. The results of assaying sphere formation when CDX1 is knocked down using hCDX1 shRNA are shown. The upper part of FIG. 14 shows the result of morphology observation, and the lower left part shows the result of comparing the amount of transcription of CD133. The lower right is the number of spheres formed. The result of having investigated CD133 expression of the neuroblastoma cell using FACS is shown. It was confirmed that CDX1 expression promotes CD133 expression. The result of having investigated the influence with respect to cell proliferation of FLAG-hCDX1 overexpression is shown. The result of having investigated the influence with respect to tumor growth of CDX1 expression is shown. The result of having investigated the influence with respect to the tumor primary culture cell of CDX1 and CD133 is shown. The result of having averaged the influence with respect to the tumor primary culture cell of CDX1 and CD133 is shown. The result which confirmed that CD133 partially canceled the influence in the formation of the sphere at the time of knocking down of CDX1 is shown. The result of having investigated the influence with respect to the expression of a cell reprogramming factor using a CDX1 modified cell is shown. It was confirmed that CDX1 promotes transcription of OCT4. The ChIP assay results are shown. CDX1 was confirmed to act by directly binding to promoters A and D of OCT4.

1. Determination of the malignancy of neuroblastoma The present inventors have shown that the transcription amount of CDX1 is increased in the neuroblastoma sphere (see FIG. 8), and that cell proliferation is promoted when CDX1 is overexpressed (FIG. 8). 16) and CDX1 expression was found to promote tumor growth (FIG. 17). On the contrary, it was also confirmed that when CDX1 was knocked down, neuroblastoma sphere formation was suppressed (FIG. 14). Thus, the expression level of the CDX1 gene is associated with the promotion of neuroblastoma sphere formation and thus the malignancy of neuroblastoma. From these facts, it is possible to determine the malignancy of neuroblastoma by measuring the expression level of the CDX1 gene. CDX1 is a transcription factor with a homeobox that recognizes and binds to a specific sequence on the genome, particularly the core sequence TTTAT (Nucleic Acids Res. 2003 Sep 15; 31 (18): 5238-46) . Since the number of vertebrae and the like in CDX1 knockout mice increases and decreases, it has been suggested that it is important for the formation of somites such as bones in the development of living bodies (Cell. 1995 Nov 17; 83 (4): 641- 53; Development. 2002 May; 129 (9): 2181-93). In addition, due to the balance of the expression level with CDX2, which is the same CDX family, it has recently become clear that it works for carcinogenesis in the stomach and small intestine and in cancer suppression in the large intestine (Proc Natl Acad Sci US A. 2012 Dec 11; 109 (50): 20584-9; Oncogene. 2008 Jul 24; 27 (32): 4497-502; Int J Cancer. 1997 Feb 20; 74 (1): 35-44; Oncogene. 2003 Sep 11; 22 (39): 7913-21 etc.).

  That is, the present invention provides a method for determining the malignancy of a neuroblastoma in a subject or a method for assisting the determination of the malignancy. The method includes the steps of (a) determining the expression level of the CDX1 gene in a sample obtained from the subject, and (b) comparing the determined expression level of the CDX1 gene with a standard. When the expression level of the CDX1 gene is higher than the standard, it is determined that the malignancy of the neuroblastoma is high or assists in the determination.

  As used herein, the degree of malignancy of neuroblastoma refers to the degree of malignancy of various properties such as metastatic / recurrent and proliferation rate of neuroblastoma. The higher the malignancy of neuroblastoma, the more likely it is to metastasize and recur, making treatment difficult. Conversely, the lower the malignancy of neuroblastoma, the easier it is to treat, and no treatment is required, or spontaneous regression may be seen in some cases, and metastasis and recurrence are rare. In the present specification, high-grade neuroblastoma is sometimes referred to as high-risk neuroblastoma, and low-grade neuroblastoma is sometimes referred to as low-risk neuroblastoma.

  As used herein, determining the grade of neuroblastoma means determining whether the grade of neuroblastoma is high or low. By using the method according to the present invention, the malignancy of neuroblastoma can be determined only by an in vitro process without intervention of a doctor.

  Since the expression level of the CDX1 gene is associated with the promotion of neuroblastoma sphere formation, and hence the malignancy of neuroblastoma, the method according to the present invention is used for the prognosis determination of neuroblastoma or for the aid of the prognosis determination. You can also. The method includes (a) determining the expression level of the CDX1 gene in a sample obtained from the subject, and (b) comparing the determined expression level of the CDX1 gene with a standard. When the expression level of the CDX1 gene is higher than the standard, it is determined that the prognosis is poor or assists in the determination.

  In this specification, “good prognosis” for neuroblastoma means that the neuroblastoma is localized or has spontaneously regressed into a benign tumor, and the possibility of metastasis or recurrence of neuroblastoma Refers to a condition with little or no or little need for treatment. In general, the prognosis is good when the grade of neuroblastoma is low.

  In the present specification, “prognosis is poor” for neuroblastoma refers to a state in which progression of a neuroblastoma tumor is recognized and malignancy is judged to be high from known diagnostic markers. In general, when the grade of neuroblastoma is high, the risk of recurrence and metastasis is high, and the prognosis is poor.

  In the present specification, a gene includes not only a structural gene or exon corresponding to a transcription product but also a regulatory region such as a promoter, a non-transcribed region, and a non-translated region such as an intron. Therefore, the expression level of a gene means the transcription amount of the gene or the expression level of the protein encoded by the gene when the gene encodes a protein, and is encoded by the amount of the transcription product (mRNA) or the gene. It can be determined by measuring the expression level of the protein. A gene expression level higher than the standard means that there is a statistically significant difference when compared to the standard, and the expression level of the gene is significantly higher. A person skilled in the art can appropriately determine a statistically significant difference from the standard deviation or standard error. Moreover, those skilled in the art can appropriately determine a low expression level when compared with a standard.

  In one embodiment, the standard corresponds to the expression level of the CDX1 gene in a healthy subject or the expression level of the CDX1 gene in non-cancerous tissue of the same subject being tested. The standard expression level of the CDX1 gene can be established by routine experimentation and statistical analysis. One skilled in the art can determine a predetermined standard expression level, eg, a standard CDX1 expression level in a healthy subject, using well-known methods.

  As used herein, a healthy subject refers to a subject or subject not diagnosed with neuroblastoma or diagnosed with cancer. In another embodiment, a healthy subject refers to a subject or subject that does not have cells capable of forming neuroblastoma spheres or does not have neuroblastoma cancer stem cells.

  The method according to the present invention can be used to determine whether a sample obtained from a subject is derived from a subject having a highly malignant neuroblastoma. In this case, if the expression level of CDX1 is higher than the standard, it can be determined that the sample is derived from a subject having high-grade neuroblastoma.

2. Detection of cells capable of forming neuroblastoma spheres The present inventors have shown that the amount of transcription of the CDX1 gene is increased in the neuroblastoma sphere (FIG. 8), and that when CDX1 is knocked down, the sphere formation of the neuroblastoma occurs. It was found that it was suppressed (FIG. 14) and that CDX1 promoted sphere formation (FIGS. 18 and 19). For these reasons, neuroblastoma cells with high CDX1 gene expression levels show strong sphere-forming properties. By measuring the expression level of the CDX1 gene using this fact, cells capable of forming neuroblastoma spheres can be detected.

  That is, the present invention provides a method for detecting cells capable of forming neuroblastoma spheres in a subject. The method includes the steps of (a) determining the expression level of the CDX1 gene in a sample obtained from the subject, and (b) comparing the determined expression level of the CDX1 gene with a standard. A higher expression level of the CDX1 gene than the standard indicates the presence of cells capable of forming neuroblastoma spheres in the sample.

  The presence of neuroblastoma spheres and the presence of cancer stem cells in the neuroblastoma will be described in relation to the grade of neuroblastoma. The presence of cells capable of forming a neuroblastoma sphere increases the incidence of metastasis, and the neuroblastoma is at high risk, and the malignancy of neuroblastoma is high. In addition, when cancer stem cells are present in neuroblastoma, the neuroblastoma is likely to recur after treatment, is a high-risk neuroblastoma, and the neuroblastoma has a high degree of malignancy. Conversely, if there are no cells capable of forming neuroblastoma spheres and / or no neuroblastoma cancer stem cells, the risk of the neuroblastoma is low and the malignancy is low.

  In one embodiment, the method according to the present invention may further comprise the step of measuring the expression level of an additional biomarker to determine the grade of neuroblastoma or to determine the prognosis. Examples of such additional biomarkers include MYCN, TRKA, ALK, CD133 and the like.

  The sample obtained from the subject used in the method according to the present invention may be any sample as long as it is derived from the subject, and includes, for example, a tissue, particularly a biopsy tissue, a tumor tissue, a brain tumor tissue, and a neuroblastoma. Or a sample derived from a cell mass suspected to be a neuroblastoma sphere, body fluid, blood, serum, plasma, or the like.

  The CDX1 gene according to the present invention can be derived from mammals, humans, monkeys, mice, rats and the like. Preferably, the CDX1 gene is human.

3. Kit The present invention includes means for determining the expression level of the CDX1 gene, for determining the malignancy of neuroblastoma, for determining the prognosis of neuroblastoma, or for detecting cells capable of forming a neuroblastoma sphere Provide kits. Examples of means for determining the expression level of the CDX1 gene include a primer for detecting the expression of the CDX1 gene, a labeled nucleic acid that targets the mRNA of the CDX1 gene, or a labeled anti-CDX1 antibody. Primers for detecting the expression of the CDX1 gene can be designed so that the exon of the CDX1 gene, the mRNA sequence of the CDX1 gene, the cDNA sequence of the CDX1 gene, etc. serve as a template. The antibody may be polyclonal or monoclonal, and the antigen-binding domain of the antibody can also be used.

  The present invention also relates to the use of CDX1 in determining the grade of neuroblastoma, for the detection of cells capable of forming neuroblastoma spheres, and / or in the manufacture of a kit for assisting the prognosis of neuroblastoma. Provided is the use of a primer, a labeled nucleic acid that targets the mRNA of the CDX1 gene, or a labeled anti-CDX1 antibody.

  The kit according to the present invention may appropriately include other components such as a buffer, a preservative, and a protein stabilizer, reagents, and instructions for use.

  As a means of measuring the expression level of the CDX1 gene, any means can be used as long as it can identify the expression level of the CDX1 gene contained in biological materials such as tissues and blood. Examples include primers for detection, labeled nucleic acids that target CDX1 gene mRNA, and labeled anti-CDX1 antibodies.

As a primer for detecting the expression of the CDX1 gene, cDNA can be synthesized from a transcription product of the CDX1 gene, and the expression level of the CDX1 gene can be measured by, for example, performing quantitative PCR on the cDNA. The labeled nucleic acid targeting the CDX1 gene mRNA can be, for example, a nucleic acid that specifically hybridizes with the CDX1 gene mRNA under stringent conditions. Stringent conditions as used herein, e.g. ^{Molecular cloning, a Laboratory manual 2 nd} edition, Cold Spring Harbor Laboratory Press (J. Sambrook et al., 1989), and the conditions described. For example, 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA and complete hybrid with probe The conditions under which hybridization occurs at a temperature about 5 ° C.-30 ° C., preferably about 10 ° C.-25 ° C. lower than the melting temperature (Tm). By using a nucleic acid that specifically hybridizes with such CDX1 gene mRNA under stringent conditions, it is possible to detect, measure or quantify the CDX1 gene mRNA contained in the sample. When a labeled anti-CDX1 antibody is used, a protein encoded by the CDX1 gene can be detected. Alternatively, an unlabeled anti-CDX1 antibody and a labeled secondary antibody that binds to the antibody can also be used. Systems using such secondary antibodies are also broadly used herein and are encompassed by labeled anti-CDX1 antibodies.

4). Screening for CDX1 Inhibiting Compounds The present inventors have found that the transcription amount of CDX1 is increased in neuroblastoma spheres (see FIG. 8). In addition, it was found that overexpression of CDX1 promotes cell proliferation (FIG. 16) and that CDX1 expression promotes tumor growth (FIG. 17). On the contrary, it was also confirmed that when CDX1 was knocked down, neuroblastoma sphere formation was suppressed (FIG. 14). Therefore, inhibition of CDX1 activity suppresses the formation of neuroblastoma spheres. Using this fact, a compound that inhibits the activity of CDX1 can be screened. That is, the present invention provides a screening method for compounds that inhibit the activity of CDX1. This method
(A) providing a CDX1 gene expression product or a nucleic acid construct having a CDX1 gene and a candidate compound to a cell capable of forming a neuroblastoma sphere;
(B) culturing the cells under conditions that allow formation of neuroblastoma spheres, and when the nucleic acid construct having the CDX1 gene in (a) is used, the CDX1 gene in the nucleic acid construct can be expressed. about,
(C) counting the number of neuroblastoma spheres formed, and (d) comparing the number measured in (c) with a positive control. A positive control is a cell that can form neuroblastoma spheres under conditions that can form neuroblastoma spheres in the absence of a candidate compound and in the presence of a CDX1 gene expression product or a nucleic acid construct having a CDX1 gene and (b ) Is the number of neuroblastoma spheres formed when cultured under conditions where the CDX1 gene in the nucleic acid construct can be expressed. Inhibition of neuroblastoma sphere formation indicates that the candidate compound inhibits CDX1.

  Examples of cells that can be used in the screening method according to the present invention include various neuroblastoma cell lines such as brain tumor cell lines and hepatoblastoma cell lines. Examples of cells that can form a neuroblastoma sphere include IMR32, SMS-SAN, and NGP. Preferably, cells that have increased transcription of the CDX1 gene can be used as cells capable of forming neuroblastoma spheres. The conditions under which neuroblastoma spheres can be formed refer to culturing neuroblastoma cells under a condition containing EGF and FGF in a serum-free medium using a culture solution called SFM. See also Takenobu et al., 2011, Oncogene, 30, 97-105 (Non-patent Document 25) for culture conditions. The formation of neuroblastoma spheres can be confirmed by morphological observation in an appropriate liquid culture medium using a microscope as necessary, and the number of blastoma spheres formed can be measured. . In the present specification, the formation of neuroblastoma spheres is suppressed when cells that have been confirmed to form neuroblastoma spheres are cultured under conditions under which neuroblastoma spheres are formed (this specification The number of neuroblastoma spheres formed is small compared to this (sometimes referred to as a positive control in the document). A small number of neuroblastoma spheres formed means that there is a statistically significant difference and that there are significantly fewer observed spheres than the number of positive control spheres.

4.1 Screening of compounds that inhibit CD133 transcription promoting activity of CDX1 The present inventors have found that CDX1 promotes transcription of the CD133 gene and induces expression of the CD133 gene (FIGS. 9 and 10). Further, at that time, it was confirmed that CDX1 induces transcription of CD133 via CD133 promoter P1 (FIG. 11), and that CDX1 acts by directly binding to CD133 promoter P1 (FIG. 13). By utilizing this fact, a compound that inhibits the CD133 transcription promoting activity of CDX1 can be screened. That is, the present invention provides a method for screening for a compound that inhibits CD133 transcription promoting activity of CDX1. This method consists of the following steps:
(A) adding an expression product of the CDX1 gene and a candidate compound to a transformed cell or model non-human animal containing a nucleic acid construct having the promoter P1 of CD133 operably linked to a reporter gene;
(B) culturing or growing the cell or model non-human animal under conditions where the reporter gene can be expressed; and (c) detecting the expression of the reporter gene.
including. The absence of detection of reporter gene expression indicates that the candidate compound inhibits CDX1's CD133 transcription promoting activity.

  In one embodiment, the promoter P1 of CD133 has the base sequence of SEQ ID NO: 30 or the base sequence of SEQ ID NO: 38. SEQ ID NO: 38 is part of SEQ ID NO: 30 and has multiple putative CDX1 binding sequences. The estimation of the CDX1 binding site can be performed using known search software such as TF Search. A person skilled in the art can specify a base sequence necessary for binding of CDX1 by conducting a conventional experiment using such software, and can use it for the screening method according to the present invention.

4.2 Screening of compounds that inhibit OCT4 transcription-promoting activity of CDX1 Further, the present inventors have found that CDX1 promotes the transcription of OCT4 and induces the expression of OCT4 (FIG. 21). At this time, it was confirmed by ChIP assay that CDX1 acts directly by binding to the promoter of OCT4 (FIG. 22). By utilizing this fact, a compound that inhibits the OCT4 transcription promoting activity of CDX1 can be screened. OCT4 (OCT3 / 4, POU5F1) is a transcription factor having a homeodomain, and is the most important factor in the maintenance and production of ES cells and iPS cells. OCT4 has been shown to directly induce the expression of various ES-related genes such as Nanog and maintain the pluripotency of cells, and when the expression level of OCT4 is decreased, ES cells quickly differentiate and Loss of properties (Nat Genet. 2000 Apr; 24 (4): 372-6). In recent years, an increase in the expression of OCT4 has been reported in various cancers, suggesting an involvement in the properties of cancer stem cells (Oncogene. 2001 Dec 6; 20 (56): 8085-91; Cancer Res 2009 Mar 1; 69 (5): 1776-81;). For OCT4, see also Mol Biol Cell. 2007 May; 18 (5): 1543-1553.

That is, the present invention provides a method for screening a compound that inhibits the OCT4 transcription promoting activity of CDX1. This method consists of the following steps:
(A) adding a CDX1 gene expression product and a candidate compound to a transformed cell or model non-human animal comprising a nucleic acid construct having an OCT4 promoter operably linked to a reporter gene;
(B) culturing or growing the cell or model non-human animal under conditions where the reporter gene can be expressed; and (c) detecting the expression of the reporter gene.
including. The absence of detection of reporter gene expression indicates that the candidate compound inhibits CDX1's OCT4 transcription promoting activity.

  In certain embodiments, the promoter of OCT4 is the OCT4 promoter region. The base sequence of the hOCT4 promoter region is shown in SEQ ID NO: 37. As the OCT4 promoter, the whole or a part of the base sequence of SEQ ID NO: 37, for example, the OCT4 promoter A shown in SEQ ID NO: 32 and / or the base sequence containing OCT4 promoter D shown in SEQ ID NO: 33 or its peripheral region may be used. it can. In other words, in the present specification, the OCT4 promoter refers to all or a part of the nucleotide sequence of SEQ ID NO: 37, for example, the sequence shown in SEQ ID NO: 32 and / or the sequence shown in SEQ ID NO: 33, or a peripheral region thereof. The base sequence may be included. The peripheral region preferably includes one or more CDX1 binding sites.

  In the present specification, “operably linked” means that promoters, genes, and the like are linked to each other so as to exhibit their functions and to express the target gene. In order for promoters and genes to perform their functions, it is necessary to match the 5 'to 3' directionality in the nucleotide sequence. For example, a coding sequence is operably linked to a promoter means that the coding sequence and the promoter are linked to each other so that transcription or expression of the coding sequence is under the control or influence of the promoter. Also, when expressing the product of two genes as a fusion protein, it can be said that the two genes are functionally linked. In this case, there should be no frame shift when linking the coding sequences of the two genes and the C-terminus of the expression product of one gene is directly linked to the N-terminus of the expression product of the other gene, or It is necessary to arrange the base sequences of the respective genes so that they are linked via an appropriate linker.

  The reporter gene may be any gene as long as it can detect the expression of the target gene, and examples thereof include luciferase, fluorescent protein, and antibiotic resistance gene. Multiple reporters may be combined. Those skilled in the art can appropriately design an expression vector and a transformation technique used for transformation. The reporter gene may be located downstream of CD133 or OCT4, or may be expressed as a fusion protein with CD133 or OCT4. The reporter protein and the expression product of CD133 or OCT4 in such a fusion protein may optionally be linked via a peptide linker. Further, the reporter gene may be arranged downstream of the promoter P1 of CD133 or the promoter of OCT4, for example, promoter A or D of OCT4 so as to be operably linked to the promoter. A reporter gene can also be placed downstream of the CDX1 recognition sequence.

  The cell that can be used for screening for a compound that inhibits CD133 or OCT4 transcription promoting activity of CDX1 may be any cell suitable for transformation, such as prokaryotic cells, E. coli, eukaryotic cells, Examples include yeast, insect cells, mammalian cells and the like. Neuroblastoma cells such as IMR32, SMS-SAN, NGP and the like may also be used.

  Examples of model non-human animals that can be used in the screening method according to the present invention include, but are not limited to, mammals excluding humans, monkeys, mice, rats, etc., nematodes, and insects.

  Candidate compounds include, but are not limited to, low molecular weight compounds, peptides, polypeptides, proteins, transcription factors, fusion proteins, nucleic acids (DNA, RNA, shRNA, siRNA, etc.) and the like. For screening, compounds contained in an appropriate compound library or combinatorial library may be used. Cell culture conditions and candidate compound addition conditions can be appropriately set by those skilled in the art.

  Examples of nucleic acid constructs that can be used in the present invention include plasmid vectors and viral vectors, particularly retroviral vectors that are integrated into the host genome. In certain embodiments, the nucleic acid construct is based on a lentiviral vector. A person skilled in the art can determine a vector, a promoter, a signal sequence and the like to be used according to the purpose of screening, and arrange them appropriately to prepare a nucleic acid construct.

  In one embodiment, CDX1, CD133, CD133 promoter P1, OCT4, or OCT4 promoter A or D is human.

  The following examples are intended for illustration only and are not intended to limit the technical scope of the present invention.

  Unless otherwise specified, the materials and reagents are commercially available, or obtained or prepared according to methods commonly used in the art and procedures in known literature.

General Experimental Methods Transfection Neuroblastoma or 293T cells are seeded in 2.5 × 10 ⁵ cells (6 cm plate) or 1 × 10 ⁶ cells (10 cm plate) and the next day 2 μg (6 cm plate) or 5 μg (10 cm Plate) pcDNA3 (mock) or pcDNA3-FLAG-hCDX1 (FLAG-hCDX1) was transfected using Lipofectamin 2000 (Life Technologies). Cells were collected 48 hours after transfection and subjected to Western blot, RT-PCR, qPCR or ChIP assay.

hCDX1 detection
hCDX1 is a specific semi-quantitative RT-PCR primer (F: TCGGACCAAGGACAAGTACC (SEQ ID NO: 3), R: TGTTGCTGCTGCTGTTTCTT (SEQ ID NO: 4)), qPCR primer (F: CGGCAGGTGAAGATCTGGTT (SEQ ID NO: 5), R: GGCTGTTGCTGCTGCTGTT (SEQ ID NO: 6)).

Quantitative PCR
Briefly, quantitative PCR (qPCR) was performed with a 7500 real-time PCR system (Applied Biosystems) using SYBR (registered trademark) Premix DimerEraser (registered trademark) (Perfect Real Time) (Takara Bio). The primers used for quantitative PCR are as follows (F: forward, R: reverse).
CD133 promoter P1 A
F: TGGGAGCGAATATGGCTTTATG SEQ ID NO: 7
R: CTTAGTGTGCAGCAGTACCAATGC SEQ ID NO: 8
CD133 promoter P1 B
F: CAGCTCAGCCGGTCCAAT SEQ ID NO: 9
R: GGATCTGCCTCAGTCACTTAAAGAT SEQ ID NO: 10
OCT4 promoter A
F: GAGGATGGCAAGCTGAGAAA SEQ ID NO: 11
R: CTCAATCCCCAGGACAGAAC SEQ ID NO: 12
OCT4 promoter D
F: GTTGGGGAGCAGGAAGCA SEQ ID NO: 13
R: GGGGCAGCTCTAACCCTAAA SEQ ID NO: 14
GAPDH promoter
F: TGAGCAGTCCGGTGTCACTA SEQ ID NO: 15
R: ACGACTGAGATGGGGAATTG SEQ ID NO: 16.

RT-PCR
RT-PCR was performed according to a conventional method. Briefly, a thermal cycler was used using a recombinant Taq (Takara Bio Inc.). The primers used for RT-PCR are as follows.
hOCT4
F: AGGCGGCTTGGAGACCT SEQ ID NO: 17
R: GATGTGGCTGATCTGCTGC SEQ ID NO: 18
hNANOG
F: AGATGCCTCACACGGAGACT SEQ ID NO: 19
R: GAGTGGTTGCTCCAGGACT SEQ ID NO: 20
hKLF4
F: ACCCTGGGTCTTGAGGAAGT SEQ ID NO: 21
R: ACGATCGTCTTCCCCTCTTT SEQ ID NO: 22
hSOX2
F: CACAACTCGGAGATCAGCAA SEQ ID NO: 23
R: GTTCATGTGCGCGTAACTGT SEQ ID NO: 24
hBMI1
F: TCTGCTGATGCTGCCAATGG SEQ ID NO: 25
R: CTCCTCATACATGACATCAATCTG SEQ ID NO: 26
GAPDH
F: ACCACAGTCCATGCCATCAC SEQ ID NO: 27
R: TCCACCACCCTGTTGCTGTA SEQ ID NO: 28.

  As for the hOCT4 promoter region, Gerrard L, et al., Stem Cells. 2005; 23 (1): 124-33 and Takahashi K, et al., Cell. 2007 Nov 30; 131 (5): 861-72 Has been reported. The primers for the hOCT4 promoter ChIP assay, OCT4 ChIP A, B, C, D, E, are reported in Freberg CT et al., Mol Biol Cell. 2007 May; 18 (5): 1543-53.

Western blot analysis Western blots were performed by conventional methods. Briefly, cells were lysed in a buffer containing 1% NP-40 to prepare a cell extract. After 30 μg of the cell extract was electrophoresed in SDS-polyacrylamide gel, it was transferred to a nylon membrane. The FLAG-tagged protein was detected using an anti-FLAG-M2 antibody (Sigma Aldrich), CDX1 using an anti-CDX1 antibody (Abcam), and CD133 using a CD133 / 2 antibody (Milteny Biotech). An anti-tubulin antibody (Lab Vision) was used as an internal control.

[Example 1]
Cloning of human CDX1 The hCDX1 gene was amplified from cDNA derived from human normal colon using hCDX1 103F-EcoRI (GAATTCGTGGGCTATGTGCTGGACAAG, SEQ ID NO: 1) and hCDX1 899R (TGGGGCTATGGCAGAAACTCC, SEQ ID NO: 2). The amplified gene was ligated downstream of the vector pcDNA3-FLAG tag (pcDNA3-FLAG-hCDX1). FLAG-hCDX1 was subcloned into a lentiviral vector (pHR-SIN-CSGW) (pHR-SIN-FLAG-hCDX1).

[Example 2]
Construction of CD133 promoter vector
The CD133 promoter P1 was cloned by PCR. 16087537-16084979 region including exon1A upstream of CD133 gene of human chromosome 4 was amplified using human genomic DNA as a template, ligated upstream of the luciferase gene of pGL4.17 vector (Promega), and pGL4.17-2.5k (- 1890 +668). Using pGL4.17-2.5k as a template, pGL4.17-1.8k (-1890, +10), pGL4.17-1.1k (-1188, +10), pGL4.17-0.4k (-1188, -752 ), pGL4.17-0.7k (-755, -34), pGL4.17-0.6k (+22, +668).

  FIG. 2 shows the positional relationship of the inserts of pGL4.17-2.5k, pGL4.17-1.8k, pGL4.17-1.1k, and pGL4.17-0.6k. pGL4.17-2.5k contains promoters P1, P2, and P3. pGL4.17-1.8k contains the sequence of 1.8k upstream of exon1A of CD133 gene. pGL4.17-1.1k contains the sequence of 1.1k upstream of exon1A of CD133 gene. pGL4.17-0.6k contains promoters P2 and P3 but not P1.

  FIG. 3 shows the positional relationship of the inserts of plasmids pGL4.17-1.1k, pGL4.17-0.4k, and pGL4.17-0.7k. Plasmid pGL4.17-1.1k has a sequence of 1.1k upstream of exon1A of CD133, and plasmid pGL4.17 has a 0.7k sequence on the side close to exon1A in the 1.1k region. The plasmid pGL4.17-0.4k has a 0.4k sequence far from exon1A in the 1.1k region.

[Example 3]
Search for CD133 promoter specific to neuroblastoma spheres Neuroblastoma cells that formed spheres in serum-free medium were collected, and total RNA was extracted using ISOGEN (Nippon Gene). From 1 to 5 μg of total RNA, cDNA was synthesized using Ribatraace α (Toyobo), and RT-PCR was performed. As the primers for detecting CD133 ORF (exon 2-7) and GAPDH, Takenobu (Takenobu H et al., Oncogene. 2011 Jan 6; 30 (1): 97-105.) Was used. For detection of CD133 exon 1A, 1B, 1C, 1D and 1E, primers of Tabu et al. (Tabu K et al, Cell Res. 2008 Oct; 18 (10): 1037-46) were used.

  The results are shown in FIG. exon1A was strongly expressed in the sphere. From this, it was confirmed that CD133 expression was induced in the neuroblastoma sphere using the promoter P1 upstream of exon 1A.

[Example 4]
CD133 promoter assay The CD133 promoter assay was performed according to the following procedure. After 5 × 10 ⁴ SK-N-BE, SK-N-DZ or SH-SY5Y neuroblastoma cells were seeded in a 12-well plate and allowed to adhere overnight, each plasmid 0.4 μg, pRL-TK ( Promega) 10 ng, pcDNA3-FLAG-hCDX1 (0-100 ng) was transfected using Lipofectamine 2000 (Life Technologies). Cells were collected 48 to 72 hours after transfection, and promoter activity was quantified using a Dual-Luciferase Reporter assay kit (Promega).

  The results of the promoter assay are shown in FIGS. As shown in FIG. 5, the strongest luciferase activity was observed for the promoter P1 (1.1k). The vector used was pGL4.17 and the cells were SK-N-DZ. Furthermore, the result of analyzing the 1.1k region is shown in FIG. Neuroblastoma cells SH-SY5Y had the strongest luciferase activity from plasmid pGL4.17-0.4k having a sequence of 0.4k. Similar results were obtained with neuroblastoma cells SK-N-BE. This 0.4k region has 6 CDX1 binding sites (see FIG. 7).

[Example 5]
The expression levels of various transcription factors in neuroblastoma cell lines IMR32, SMS-SAN, and NGP were examined. Expression levels were determined by quantitative PCR (qPCR) and RT-PCR.

  The results are shown in FIG. Among various transcription factors, it was confirmed that the expression of CDX1 increased sphere-specifically. From these results, it was considered that CDX1 regulates the expression of CD133, and the effect of CDX1 transduction or knockdown was examined.

[Example 6]
Lentivirus-mediated hCD133 and hCDX1 expression or knockdown in neuroblastoma cells Expression of pHR-SIN-hCD133 and pHR-SIN-CSGW-FLAG-hCDX1 by lentivirus or expression of hCDX1 shRNA incorporated into pLKO.1 , Takenobu and Ochiai (Ochiai et al., Oncogene. 2010 May 6; 29 (18): 2681-90). Briefly, a plasmid encoding a non-proliferating lentiviral backbone and the above plasmid were co-introduced into 293T cells, and the culture supernatant containing the virus was collected 48 hours and 72 hours after the introduction. The culture supernatant was added to the target cell culture medium to infect the lentivirus. Cells infected with lentivirus expressing shRNA with puromycin resistance were selected in the presence of 1 μg / ml puromycin, and only virus-infected cells were collected.

CD133 expression in cells transduced with CDX1 was examined by qPCR and Western blot analysis. The result of qPCR is shown in FIG. Compared with mock transfection, CD133 expression was increased in CDX1-transduced cells, confirming that CDX1 induces CD133 expression.
The results of Western blot analysis are shown in FIG. The expression product of CDX1 was confirmed.

  Further, the results of examining the expression of CD133 when CDX1 is expressed are shown in FIG. For neuroblastoma cells SK-N-BE, transcription of CD133 ORF and CD133 exon1A was induced in cells transfected with CDX1 compared to when Mock transfected. This trend was similar for NGP and 293T cells.

  The results of examining the expression of CD133 when CDX1 was knocked down are shown in FIG. In the NGP sphere, knocking down CDX1 reduced CD133 expression compared to Mock transfection.

[Example 7]
ChIP assay
To examine whether CDX1 binds directly to CD133 promoter P1, a ChIP assay was performed. This ChIP assay was performed by previously reported methods (Orlando et al., 1997, Methods, 11, 205-214, Fujimura et al., 2006, Development, 133, 2371-2381). Briefly, FLAG-CDX1 was expressed in cells, and protein and DNA were cross-linked by formalin fixation. CDX1 was precipitated with antibody and chromatin immunoprecipitation was performed. The promoter P1 genome of CD133 was detected by quantitative PCR. Specific conditions and procedures are as follows.

  Cells were harvested 48 hours after transfection. Cross-linked chromatin prepared from labeled cells was precipitated with normal mouse IgG (eBioscience, San Diego, CA, USA) or monoclonal anti-Flag antibody (M2; Sigma-Aldrich). Total DNA (or input DNA) was isolated from the initial lysate. DNA extracted from each fraction was analyzed by qPCR.

  The results are shown in FIG. In ChIP (1), the value of FLAG-CDX1 was significantly higher than that of mock, confirming that CDX1 directly binds to the promoter P1 of CD133.

[Example 8]
The sphere formation assay The medium used for the sphere formation assay is as previously reported (Takenobu et al., 2011, Oncogene, 30, 97-105). Briefly, the medium is SFM, a serum-free medium containing EGF, FGF and B27 additives. Cells expressing or knocked down hCDX1 were seeded with 1 × 10 ⁵ cells in 6-well plates and 100 cells in 96-well plates, and the medium was changed every 7 days. The number of spheres was measured under a microscope 2 weeks to 1 month after medium replacement.

  hCDX1 shRNA was used to assay sphere formation when CDX1 was knocked down. The results are shown in FIG. When the relative sphere number at the time of Mock transfection was 100, the number of spheres formed when CDX1 was knocked down was less than 40 (lower right in FIG. 14). In other words, CDX1 knockdown suppressed sphere formation. It was also confirmed that the amount of CD133 transferred was reduced under these experimental conditions (lower left of FIG. 14).

[Example 9]
FACS (Fluorescence Assisted Cell Sorting) analysis
FACS analysis was performed according to the following procedure. First, neuroblastoma cells NGP were infected with a lentivirus expressing human CDX1 (a virus prepared by introducing pHR-FLAG-hCDX1), and the cells were collected one week after the virus infection. The collected cells or uninfected NGP cells were stained with PE-labeled mouse control IgG (eBioscience) or CD133 / 2 PE (clone 293C3, Miltenyi Biotec), and the intensity of PE fluorescence was analyzed with FACS Calibur (BD).
The results of examining CD133 expression in neuroblastoma cells using FACS are shown in FIG. It was also confirmed from FACS that CDX1 promotes the expression of CD133.

[Example 10]
Effects of CDX1 overexpression The experimental procedure was briefly described using the WST method and the soft agar colony formation method. In the WST method, 750 cells were seeded in a 96-well plate, and cell proliferation was examined with a microplate reader (Corona) using Cell Counting Kit-8 reagent (Dojin Chemical) every 24 hours. In soft agar colony formation, 5,000 cells were suspended in a medium containing 0.3% agar (Difco) and added to a 3.5 cm cell culture dish. Colonies were stained overnight with 0.05% MTT solution (Nacalai Tesque) three weeks after sowing, and photographs were taken to count the number of colonies.

  FIG. 16 shows the results of examining the influence on cell proliferation when FLAG-hCDX1 is overexpressed. In transformed cells (SH-SY5Y CDX1) introduced with hCDX1 compared to mock transfection, cell proliferation was promoted (FIG. 16, upper left), and the number of colonies formed on the agar medium was also compared with mock transfection (EGFP). (Fig. 16, right).

[Example 11]
Effect of CDX1 on tumor growth The experimental procedure was simply to introduce cells subcutaneously into nude mice. The experimental animals used were BALB / c Ajcl-nu / nu, female, 5 weeks old, and the vector was pHR-SIN-CSGW-FLAG-hCDX1.

The results of examining the effect of CDX1 expression on tumor growth are shown in FIG. With mock transfection (GFP), the tumor size was 1000 mm ³ 40 days after inoculation, whereas when CDX1 was overexpressed, the tumor size was about 2000 mm ³ 40 days after inoculation. there were.

[Example 12]
Effect of CDX1 or CD133 on primary cultured cells of tumors Cultured cells were transduced with CDX1 or CD133. 1 × 10 ⁶ cells (spheres) were infected with lentivirus (enriched with lenti-X, serum-free, × 2). After incubation for 2 days, the infection was confirmed by GFP positive, and the cells were dissociated by pipetting. Viable cells were counted and 100 cells were seeded in each well using a 96 well plate. This was incubated for 11 days and then observed. The cells used were 2209BM, 2302BM, 2338BM.

  FIG. 18 shows the results of examining the effect of CDX1 or CD133 on primary cultured cells of the tumor. Both CDX1 and CD133 promoted sphere formation. Moreover, what averaged these values is shown in FIG.

[Example 13]
Effects of CDX1 knockdown
CDX1 expression was knocked down using shRNA technique. The cells used were NGP or IMR32. The sequences of the shRNA for CDX1 knockdown used are shown in SEQ ID NO: 35 and SEQ ID NO: 36. In addition, what is shown by sequence number 35 and 36 is the DNA sequence integrated via the lentivirus, and the RNA sequence synthesize | combined based on the DNA sequence gives knockdown as shRNA. FIG. 20 shows the result of confirming that CD133 partially cancels the influence on the sphere formation when CDX1 is knocked down.

[Example 14]
Cell reprogramming factor gene expression in CDX1-modified cells
CDX1 modified cells were used to examine the effect of CDX1 on the expression of cellular reprogramming factors. The cell lines used for overexpression were SH-SY5Y, SK-N-BE, and 293T, and the sphere cells used for knockdown were NGP (1), NGP (2), and Huh6. Cells were expressed FLAG-CDX1 to overexpress CDX1. In addition, shRNA was used for CDX1 knockdown. These modified cells were detected by semi-quantitative RT-PCR for the expression of molecules related to stem cells, including Yamanaka four factors.
The results are shown in FIG. CDX1 was confirmed to promote OCT4 transcription.

  Further, FIG. 22 shows the result of ChIP assay for examining the binding of CDX1 to the OCT4 promoter. CDX1 was confirmed to bind directly to promoter site A or D of OCT4. The experimental conditions were almost the same as above except that CD133 was replaced with OCT4.

  By measuring the expression level of the CDX1 gene in a sample obtained from a subject, the grade of malignancy of the subject's neuroblastoma can be determined, and the prognosis can be determined. Furthermore, by screening for compounds that inhibit CDX1 activity, candidate compounds that can be therapeutic agents for neuroblastoma can be identified.

SEQ ID NOs: 1-28 As in Examples.
SEQ ID NO: 29hCD133
SEQ ID NO: 30 hCD133 promoter P1
SEQ ID NO: 31hOCT4
SEQ ID NO: 32 hOCT4 promoter A
SEQ ID NO: 33 hOCT4 promoter D
SEQ ID NO: 34hCDX1
SEQ ID NO: 35 shRNA for CDX1 knockdown, shCDX1 A
SEQ ID NO: 36 shRNA for CDX1 knockdown, shCDX1 B
SEQ ID NO: 37 promoter region of hOCT4 SEQ ID NO: 38 Partial sequence of promoter P1 of hCD133

Claims (8)

A way to help detect high cell proliferative neuroblastoma cells,
(A) determining the expression level of the CDX1 gene in a sample containing neuroblastoma cells obtained from a subject;
(B) comparing the determined CDX1 gene expression level with a standard;
Hints, wherein CDX1 gene expression level is an auxiliary that you determined to be higher than the standard high cell proliferative neuroblastoma cells, said method. A method of detecting neuroblastoma cells capable of forming a neuroblastoma sphere in a subject comprising:
(A) determining the expression level of the CDX1 gene in a sample containing neuroblastoma cells obtained from a subject;
(B) comparing the determined CDX1 gene expression level with a standard;
Wherein the expression level of the CDX1 gene in the sample is higher than the standard indicates the presence of neuroblastoma cells capable of forming neuroblastoma spheres in the subject. Comprising means for determining the expression level of the CDX1 gene to detect cell proliferation highly neuroblastoma, or a kit for the detection of neuroblastoma cells which can form a neuroblastoma spheres, wherein the CDX1 gene The kit described above, wherein the means for determining the expression level is a primer for detecting the expression of the CDX1 gene, or a labeled nucleic acid that targets the mRNA of the CDX1 gene . A method for screening a compound that inhibits CDX1, comprising the following steps:
(A) CDX1 gene and expression product protein or nucleic acid construct having a CDX1 gene candidate compound, introduced into a cell capable of forming a neuroblastoma spheres,
(B) the cells are cultured under conditions capable of forming a neuroblastoma sphere, and when the nucleic acid construct having a CDX1 gene in (a) is used, the CDX1 gene in the nucleic acid construct can be expressed. ,
(C) counting the number of neuroblastoma spheres formed, and (d) comparing the number measured in (c) above with a positive control,
A positive control is a cell that can form neuroblastoma spheres under conditions that can form neuroblastoma spheres in the absence of a candidate compound and in the presence of a CDX1 gene expression product or a nucleic acid construct having a CDX1 gene and (a ) Is the number of neuroblastoma spheres formed when cultured under conditions where the CDX1 gene in the nucleic acid construct can be expressed,
Said method, wherein the formation of neuroblastoma spheres is suppressed compared to a positive control, indicating that the candidate compound inhibits CDX1. A method for screening a compound that inhibits CD133 transcription-promoting activity of CDX1, comprising the following steps:
(A) adding an expression product protein of the CDX1 gene and a candidate compound to a transformed cell or model non-human animal containing a nucleic acid construct having the promoter P1 of CD133 operably linked to a reporter gene;
(B) culturing or growing the cell or model non-human animal under conditions where the reporter gene can be expressed; and (c) detecting the expression of the reporter gene.
Wherein the reporter gene expression is not detected indicates that the candidate compound inhibits CD133 transcription-promoting activity of CDX1. The method according to claim 5 , wherein the promoter P1 of CD133 has the nucleotide sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 38. A method for screening a compound that inhibits the OCT4 transcription-promoting activity of CDX1, comprising the following steps:
(A) adding a CDX1 gene expression product protein and a candidate compound to a transformed cell or model non-human animal comprising a nucleic acid construct having an OCT4 promoter sequence operably linked to a reporter gene;
(B) culturing or growing the cell or model non-human animal under conditions where the reporter gene can be expressed; and (c) detecting the expression of the reporter gene.
Hints, the expression of the reporter gene is not detected, it indicates that the candidate compound inhibits OCT4 transcription promoting activity of CDX1, but the promoter sequences of the OCT4 is having the sequence of the promoter A and / or D of OCT4 The method, wherein OCT4 promoter A has the base sequence set forth in SEQ ID NO: 32 and / or OCT4 promoter D has the base sequence set forth in SEQ ID NO: 33 . CDX1 gene is a human CDX1 gene having the nucleotide sequence set forth in SEQ ID NO: 34, The method according to any one of claims 4-7.

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