Method for screening material for inhibiting exacerbation of glioblastoma cells

Abstract

The present disclosure provides a means for screening a material for inhibiting the exacerbation of glioblastoma cells. The present inventors have found the possibility of solving the problem by means of a method for screening a material having an exacerbation inhibiting action against glioblastoma cells, the method comprising a step for evaluating the Ror1 signaling inhibiting action of a test substance and / or a step for evaluating the Ror2 signaling inhibiting action of the test substance, thereby completing the present disclosure.

Description

Screening method for substances that inhibit the progression of glioblastoma cells

[0001] The present disclosure relates to a screening method for a substance that suppresses the progression of glioblastoma cells, and the like.

[0002] Gliomas, which are brain tumors, are classified into grades 2 to 4 according to their malignancy, with the most malignant grade 4 gliomas being called glioblastomas. The five-year survival rate for glioblastomas is less than 10%, making for an extremely poor prognosis. Glioblastomas grow rapidly, are resistant to radiation and anticancer drugs, and have a high rate of recurrence, so treatment outcomes with multidisciplinary treatment have not improved for a long time.

[0003] In recent years, it has become clear that glioblastoma cells contain cells with undifferentiated stem cell-like properties, which contribute to the progression of glioblastoma.

[0004] Cell Biochem Funct. 2016 Apr;34(3):149-57. doi: 10.1002 / cbf.3172.Cancer Sci. 2023 Feb;114(2):561-573. doi: 10.1111 / cas.15630.Cell Discov. 2022 Oct 5;8(1):101. doi: 10.1038 / s41421-022-00445-8.PLoS One. 2017; 12(3): e0170675. doi: 10.1371 / journal.pone.0170675Redox Biol. 2019 May;23:101107. doi: 10.1016 / j.redox.2019.101107.

[0005] Previous studies have reported that Ror1 protein, a member of the Ror family of receptors, is highly expressed in glioblastoma cells and that Ror1 signaling is involved in regulating their stemness (Non-Patent Documents 1 and 2). However, the downstream signaling of Ror1 involved in glioblastoma progression has not been elucidated, and the molecular mechanisms contributing to glioblastoma progression, such as its treatment resistance, remain largely unknown. Meanwhile, research by the present inventors has suggested that Ror2 protein, another member of the Ror family of receptors, is also highly expressed in glioblastoma, suggesting that Ror2 signaling is also involved in glioblastoma progression. Furthermore, among the Wnt family proteins that may act as ligands for Ror1 and Ror2 proteins, Wnt5a protein has been reported to be highly expressed in glioblastoma patients and correlated with poor prognosis (Non-Patent Document 2). However, it remains unclear which of the 19 Wnt family proteins contributes to glioblastoma progression through activation of Ror1 and Ror2 signaling. It is expected that elucidating these facts will lead to the development of therapeutic drugs or treatment methods for glioblastoma.An objective of the present disclosure is to provide a screening method for substances that suppress the progression of glioblastoma cells.

[0006] The present inventors have found that suppressing the expression of the Ror1 gene in glioblastoma cells reduces the expression of the Pla2g2a gene and suppresses the stemness of the glioblastoma cells. Furthermore, the present inventors have found that suppressing the expression of the Ror2 gene in glioblastoma cells reduces the level of Nrf2 in the cell nucleus and reduces the expression of genes that suppress ferroptosis. Furthermore, the present inventors have found that the expression levels of genes encoding the Wnt10a protein and the Wnt16 protein are high in glioblastoma cells, and have made further improvements.

[0007] The present disclosure encompasses, for example, the subject matter described in the following paragraphs: Item 1. A method for screening for a substance having an inhibitory effect on the progression of glioblastoma cells, comprising the steps of: evaluating the inhibitory effect of Ror1 signaling of a test substance; and / or evaluating the inhibitory effect of Ror2 signaling of the test substance, wherein the inhibitory effect of Ror1 signaling is confirmed by: (A) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-a) Ror1 protein-expressing cells, and then confirming that expression of Pla2g2a gene is suppressed in Ror1 protein-expressing cells; (B) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-b) Ror1 protein, and then (C) a step of confirming that the test substance inhibits the binding of a Wnt protein and a Ror1 protein; and (D) a step of confirming that activation of Nrf2 protein in Ror2 protein-expressing cells is suppressed after contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57; (E) (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein;(ii) a step of contacting the test substance and (iv-b) a Ror2 protein, and then confirming that the test substance inhibits the binding of the Wnt protein and the Ror2 protein; (F) a step of contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-a) a Ror2 protein-expressing cell, and then contacting the Ror2 protein-expressing cell with a ferroptosis-inducing agent, and then measuring the viability of the Ror2 protein-expressing cell; and (G) a step of contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58, and then and confirming that the test substance inhibits binding between a Wnt protein and a polypeptide comprising the amino acid sequence of SEQ ID NO: 58. Item 2. The screening method according to Item 1, comprising the steps of evaluating the Ror1 signaling inhibitory activity of the test substance, and evaluating the Ror2 signaling inhibitory activity of the test substance. Item 3. The screening method according to Item 1 or 2, wherein the Ror1 signaling inhibitory activity is evaluated by a method comprising the steps of: (A) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-a) Ror1 protein-expressing cells, and then confirming that expression of Pla2g2a gene is suppressed in the Ror1 protein-expressing cells. Item 4. (D) the Ror2 signaling inhibitory effect is detected by contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iv-a) Ror2 protein-expressing cells, and thenItem 5. The screening method according to Item 1 or 2, wherein the inhibitory effect of Ror1 signaling is evaluated by a method comprising the step of: (C) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iii-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57, and then confirming that the test substance inhibits binding between the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57. Item 6. Item 7. The screening method according to Item 1 or 2, wherein the inhibitory effect of Ror2 signaling is evaluated by a method comprising the step of: (G) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iv-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58, and then confirming that the test substance inhibits binding between the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58. Item 7. A method for screening for a substance that suppresses the progression of glioblastoma cells, comprising screening for a substance that suppresses ferroptosis resistance of glioblastoma cells and / or screening for a substance that suppresses stemness of glioblastoma cells. Item 9. The method according to Item 1, wherein the method for screening for a substance that suppresses ferroptosis resistance in glioblastoma cells comprises the following steps: (α-1) contacting (I) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (II) a test substance, and (III) Ror2 protein-expressing cells, and (β-1) confirming that activation of Nrf2 protein in Ror2 protein-expressing cells is suppressed.Item 10. The method of any one of Items 7 to 9, wherein the method for screening for a substance that suppresses ferroptosis resistance in glioblastoma cells further comprises the step of: (γ-1) confirming that the test substance inhibits the binding of the Wnt protein and the Ror2 protein. Item 10. The method of any one of Items 7 to 9, wherein the method for screening for a substance that suppresses stemness in glioblastoma cells comprises the following steps: (α-2) contacting (I) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (II) a test substance, and (III) a cell expressing Ror1 protein, and (β-2) confirming that expression of Pla2g2a gene is suppressed in the cell expressing Ror1 protein. Item 11. The method of Item 10, wherein the method for screening for a substance that suppresses stemness in glioblastoma cells further comprises the step of: (γ-2) confirming that the test substance inhibits the binding of the Wnt protein and the Ror1 protein. Item 12. Item 12. A method according to any one of Items 7 to 11, wherein the method for screening for a substance that suppresses ferroptosis resistance in glioblastoma cells comprises the following steps: (α-3) contacting (I) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (II) a test substance, and (III) Ror2 protein, and (β-3) confirming that the test substance inhibits binding between the Wnt protein and Ror2 protein. Item 14. A method according to any one of Items 7 to 14, wherein the method for screening for a substance that suppresses stemness of glioblastoma cells comprises the following steps: (α-4) contacting (I) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (II) a test substance, and (III) a Ror1 protein, and (β-4) confirming that the test substance inhibits binding between the Wnt protein and the Ror1 protein.Item 14. The method according to any one of Items 7 to 13, wherein the method for screening for a substance that suppresses ferroptosis resistance in glioblastoma cells comprises the following steps: (α-5) contacting (I) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (II) a test substance, and (III) Ror2 protein-expressing cells; (β-5) contacting the Ror2 protein-expressing cells with a ferroptosis-inducing agent; and (γ-5) measuring the viability of the Ror2 protein-expressing cells.

[0008] A means for screening for a substance that suppresses the progression of glioblastoma cells (more specifically, for example, a substance that suppresses the ferroptosis resistance of glioblastoma cells and / or a substance that suppresses the stemness of glioblastoma cells) is provided.

[0009] Figure 1 shows the results of expression analysis of various genes in T98G cells (upper row) and U251MG cells (lower row) cultured in adherent or suspension culture. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, ***P<0.001. Figure 1 shows the results of expression analysis of various genes when T98G cells that formed spheroids in suspension culture were treated with strictinin (final concentration 50 μM), a Ror1 inhibitor. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, *P<0.05, **P<0.01, ***P<0.001. Figure 1 shows the expression analysis of various genes when Ror1 was forced to be expressed in T98G cells cultured in adhesion culture. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, *P<0.05, **P<0.01, ***P<0.001. Figure 1 shows the results of the expression analysis of various genes when Pla2g2a gene expression was knocked down using Pla2g2a siRNA in T98G cells that formed spheroids in suspension culture. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, **P<0.01, ***P<0.001. Figure 1 shows the results of expression analysis of various genes when adherently cultured T98G cells were stimulated with recombinant PLA2G2A protein. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, *P<0.05, **P<0.01. Figure 1 shows the results of bioinformatics analysis and survival curve analysis using the TCAG database. Figure 1 shows the results of expression analysis of the Ror2 gene in adherently cultured or suspension cultured T98G and U251MG cells. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, ***P<0.001. 1 shows the results of analyzing the relative cell proliferation ability and the relative amount of Nrf2 protein localized in the nucleus when the expression of the Ror2 gene in U251MG cells was knocked down by siRNA.Data are presented as mean ± standard deviation (S.D.). One-way ANOVA with Dunnett's test, **P<0.01, ***P<0.001. This figure shows the results of an analysis of the expression of Nrf2 target genes when the expression of the Nrf2 gene and the Ror2 gene in U251MG cells was knocked down by siRNA. Data are presented as mean ± standard deviation (S.D.). One-way ANOVA with Dunnett's test, *P<0.05, **P<0.01, ***P<0.001. This figure shows the results of an analysis of the effect of suppressing the expression of the Ror2 gene in U251MG cells on erastin (a ferroptosis inducer)-induced cell death. This figure shows the results of bioinformatics analysis and survival curve analysis using the TCAG database. Figure 1 shows the results of expression analysis of the Wnt5a gene, Wnt10a gene, and Wnt16 gene in T98G and U251MG cells cultured in adherent or suspension culture. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, **P<0.01, ***P<0.001. Figure 1 shows the results of quantitative RT-PCR analysis of the effect on various gene expression when Ror1 expression in T98G cells that formed spheroids in suspension culture was knocked down using two types of Ror1 siRNA (#1 and #2) with different sequences. Data are shown as mean ± standard deviation (S.D.). One-way ANOVA with Dunnett's test, ***P<0.001. The figures show the results of quantitative RT-PCR analysis of the effect on various gene expression when Wnt5a expression in T98G cells that formed spheroids in suspension culture was knocked down using two types of Wnt5a siRNA (#1 and #2) with different sequences. Data are shown as mean ± standard deviation (S.D.). One-way ANOVA with Dunnett's test, ***P<0.001. (Left) Schematic diagram of the entire Ror and Ror-Fc. (Right) Results of protein binding analysis (pull-down assay) of Ror1-Fc or Ror2-Fc with Wnt5a or Wnt16.Figure 1 shows the results of a protein binding analysis (pull-down assay) of Ror1-Fc, Ror1ΔCRD-Fc, Ror2-Fc, or Ror2ΔCRD-Fc with Wnt16. (Left) Schematic diagram of whole Ror1 and Ror1 decoy. (Right) Results of an analysis of the effect on Pla2g2a expression and stemness when the extracellular domain of Ror1 is forcibly expressed as Ror1 decoy in spheroid-forming T98G cells. Data are shown as mean ± standard deviation (S.D.). Unpaired two-tailed Student's t test, **P<0.01, ***P<0.001. Results of an analysis of the effect on spheroid growth rate when spheroid-forming T98G cells are stimulated with purified Wnt16 protein are shown. Data are shown as mean ± standard deviation (S.D.). Multiple unpaired two-tailed Student's t test, **P<0.01, ***P<0.001. (Left) Shows the results of analyzing the effect on RSL3 (a ferroptosis inducer)-induced cell death when U251MG cells were stimulated with purified Wnt5a protein or purified Wnt16 protein. (Right) Shows the results of analyzing the effect on RSL3-induced cell death when U251MG cells were stimulated with culture supernatant (Wnt10a CM) of Wnt10a-expressing Cos7 cells. Each data is shown as mean ± standard deviation (S.D.). One-way ANOVA with Dunnett's test, *P<0.05. (Left) The Ror1 signaling pathway and Ror2 signaling pathway inferred from the present disclosure are shown. (Right) The screening strategy for substances discovered in the present disclosure that have an inhibitory effect on the progression of glioblastoma cells is shown. The amino acid sequence of human Ror1 protein (SEQ ID NO: 59) is shown. The amino acid sequence of human Ror2 protein (SEQ ID NO: 60) is shown.

[0010] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses a screening method for a substance that suppresses the progression of glioblastoma cells, the screening method including a step of evaluating the inhibitory effect of a test substance on Ror1 signaling and / or a step of evaluating the inhibitory effect of the test substance on Ror2 signaling. This screening method may be referred to as the "screening method of the present disclosure."

[0011] Ror (Receptor tyrosine kinase-like orphan receptor) proteins are single-pass transmembrane receptor proteins that function as receptors for Wnt proteins and are known to activate the non-canonical Wnt pathway. Examples of Ror proteins include Ror1 and Ror2. For example, the accession number for the mRNA sequence of the human Ror1 gene is NM_005012, and the accession number for the mRNA sequence of the human Ror2 gene is NM_004560. Furthermore, the accession numbers for each of the mRNA sequences are listed on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ). By searching for the above, the amino acid sequence of human Ror1 protein (SEQ ID NO: 59) and the amino acid sequence of human Ror2 protein (SEQ ID NO: 60) can be obtained.

[0012] The experiments described in the Examples below suggested the existence of the following Ror1 signaling pathway in glioblastoma cells, which is initiated by the binding of at least one Wnt protein selected from the group consisting of Wnt10a, Wnt16, and Wnt5a to Ror1: Wnt protein binds to Ror1; Ror1 intracellular signaling is activated; and Pla2g2a expression level increases. Furthermore, the present inventors found that an increase in Pla2g2a expression level enhances the stemness of glioblastoma cells and exacerbates glioblastoma ( FIG. 20 ). That is, the present inventors found that inhibiting Ror1 signaling can suppress the exacerbation of glioblastoma cells.

[0013] In the present disclosure, the term "Ror1 signaling inhibitory effect" refers to an effect of inhibiting at least one of the steps constituting the Ror1 signaling pathway described above. That is, in the present disclosure, the term "Ror1 signaling inhibitory effect" refers to at least one effect selected from the group consisting of an effect of inhibiting the binding of a Wnt protein to an Ror1 protein, an effect of inhibiting an increase in the expression level of Pla2g2a, and an effect of inhibiting the enhancement of stemness of glioblastoma cells.

[0014] Furthermore, the experiments described in the Examples below suggested the existence of the following Ror2 signaling pathway in glioblastoma cells, which is initiated by the binding of at least one Wnt protein selected from the group consisting of Wnt10a, Wnt16, and Wnt5a to Ror2: Wnt protein binds to Ror2; Ror2 intracellular signaling is activated; Nrf2 protein is activated; and the expression levels of Nrf2 target genes, such as Slc7a11 and Ftl, increase. Furthermore, the present inventors found that the increased expression levels of Nrf2 target genes enhances the resistance to ferroptosis in glioblastoma cells and exacerbates glioblastoma disease ( FIG. 20 ). That is, the present inventors found that inhibiting Ror2 signaling can suppress the exacerbation of glioblastoma cell progression.

[0015] In the present disclosure, the term "Ror2 signaling inhibitory effect" refers to an effect of inhibiting at least one of the steps constituting the Ror2 signaling pathway described above. That is, in the present disclosure, the term "Ror2 signaling inhibitory effect" refers to at least one effect selected from the group consisting of an effect of inhibiting the binding of Wnt protein to Ror2 protein, an effect of inhibiting the activation of Nrf2 protein, an effect of inhibiting an increase in the expression level of target genes of Nrf2 protein such as Slc7a11 and / or Ftl, and an effect of inhibiting the enhancement of ferroptosis resistance in glioblastoma cells.

[0016] As described above, both the Ror1 signaling pathway and the Ror2 signaling pathway are involved in the progression of glioblastoma cells in parallel. Therefore, inhibiting only one of Ror1 signaling and Ror2 signaling can suppress the progression of glioblastoma cells, but inhibiting both Ror1 signaling and Ror2 signaling is expected to more effectively suppress the progression of glioblastoma cells. Therefore, it is particularly preferable that the screening method of the present disclosure includes both a step of evaluating the Ror1 signaling inhibitory effect of the test substance and a step of evaluating the Ror2 signaling inhibitory effect of the test substance.

[0017] The inhibitory effect of a test substance on Ror1 signaling can be confirmed, for example, by: (A) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-a) Ror1 protein-expressing cells, and then confirming that the expression of Pla2g2a gene in the Ror1 protein-expressing cells is suppressed; (B) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-b) Ror1 protein, and then confirming that the test substance inhibits the binding of the Wnt protein and the Ror1 protein; and (C) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) contacting the test substance with (iii-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57, and then confirming that the test substance inhibits the binding between a Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57.

[0018] The inhibitory effect of a test substance on Ror1 signaling may be evaluated by combining two or more steps from steps (A) to (C). For example, step (B) may be performed before or after step (A).

[0019] The inhibitory effect of a test substance on Ror2 signaling can be confirmed, for example, by: (D) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-a) Ror2 protein-expressing cells, and then confirming that activation of Nrf2 protein in Ror2 protein-expressing cells is suppressed; (E) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-b) Ror2 protein, and then confirming that the test substance inhibits binding of the Wnt protein and Ror2 protein; (F) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) contacting the test substance with (iv-a) Ror2 protein-expressing cells, and then contacting the Ror2 protein-expressing cells with a ferroptosis-inducing agent, and then measuring the viability of the Ror2 protein-expressing cells; and (G) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance with (iv-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58, and then confirming that the test substance inhibits binding between the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58.

[0020] The inhibitory effect of a test substance on Ror2 signaling may be evaluated by combining two or more steps from steps (D) to (G). For example, step (E) may be performed before or after step (D).

[0021] (i) Wnt Proteins Wnt (Wingless-type MMTV integration site family) proteins are secreted glycoproteins known to bind to Ror proteins and the like. Nineteen types of Wnt proteins have been identified in mammals, including Wnt10a, Wnt16, and Wnt5a. For example, the accession number for the mRNA sequence of the human Wnt10a gene is NM_025216, the accession number for the mRNA sequence of the human Wnt16 gene is NM_057168, and the accession number for the mRNA sequence of the human Wnt5a gene is NM_003392. In the screening method of the present disclosure, at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein is used.

[0022] The Wnt proteins used in the screening method of the present disclosure may be one type alone, a combination of two types, or a combination of three types. For example, they may be Wnt10a protein, Wnt16 protein, or Wnt5a protein; Wnt10a protein and Wnt16 protein; Wnt10a protein and Wnt5a protein; Wnt16 protein and Wnt5a protein; or Wnt10a protein, Wnt16 protein, and Wnt5a protein.

[0023] (ii) Test Substance The test substance is not particularly limited. For example, it may be a compound or a composition. The compound may be, for example, a low molecular weight compound, a nucleic acid (e.g., DNA, RNA, etc.), a protein (e.g., an antibody or a portion thereof, etc.), or a polymeric compound such as a polymer. The composition may be, for example, an extract obtained from an organism (e.g., an animal, a plant, a microorganism, etc.), or may be a combination of two or more compounds.

[0024] (iii-a) Ror1 Protein-Expressing Cells Ror1 protein-expressing cells are not particularly limited as long as they express Ror1 protein. For example, they may be glioblastoma cells (e.g., T98G cells or U251MG cells) or cells in which Ror1 protein is forcibly expressed (e.g., HEK293 cells). Furthermore, Ror1 protein-expressing cells may be, for example, primary culture cells, passaged cells, or established cell lines.

[0025] (iii-b) Ror1 Protein In the screening method of the present disclosure, the Ror1 protein may be, for example, a Ror1 protein expressed in Ror1 protein-expressing cells such as glioblastoma cells, as is, or may be a Ror1 protein purified by a known method from cells in which Ror1 protein is forcibly expressed. Alternatively, a Ror1 protein obtained by in vitro translation in a cell-free system may be used. Specifically, the Ror1 protein of the present disclosure includes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 59, and a protein consisting of an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 59 and that can interact with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, and more preferably 95% or more.

[0026] The "identity" of an amino acid sequence refers to the degree of amino acid sequence match between two or more comparable amino acid sequences. Therefore, the higher the identity between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. It can also be determined using a program called BLASTX, which is based on the BLAST algorithm. Specific techniques for these analysis methods are known, and can be found on the NCBI website (http: / / www.ncbi.nlm.nih.gov / ). Examples of amino acid sequences that are not 100% identical to a given amino acid sequence X include amino acid sequences in which one or more amino acids have been substituted, deleted, added, or inserted (preferably by addition or substitution, more preferably by addition or conservative substitution) relative to the amino acid sequence X. Here, "multiple" refers to, for example, 2 to 100, preferably 2 to 50, more preferably 2 to 20, even more preferably 2 to 10, and particularly preferably 2 to 5. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0027] Furthermore, whether a protein interacts with another protein can be confirmed by methods known in the art. Such methods include, for example, co-immunoprecipitation (Co-IP), pull-down assay, Western blotting, cross-linking, label transfer, interaction mapping, surface plasmon resonance, and FRET (fluorescence resonance energy transfer). Furthermore, whether two proteins interact with each other can be predicted by computer simulation based on the amino acid sequences of the proteins.

[0028] The Ror1 protein of the present disclosure includes a protein consisting of an amino acid sequence in which 1 to 100 (preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5) amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence of SEQ ID NO: 59, and which is capable of interacting with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. The Ror1 protein may be bound to or fused with any other peptide, oligopeptide, or protein, so long as it is capable of interacting with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. Examples of other peptides, oligopeptides, or proteins include albumin (e.g., serum albumin), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag), fluorescent proteins (e.g., GFP, BFP, CFP, YFP, RFP), luminescent proteins (e.g., luciferase), secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression-enhancing sequences, solubilization sequences, and multimerization domains.

[0029] (iii-c) Polypeptide Comprising the Amino Acid Sequence Represented by SEQ ID NO: 57 The amino acid sequence represented by SEQ ID NO: 57 is the amino acid sequence constituting the cysteine-rich domain (sometimes referred to as "CRD" in the present disclosure) of the Ror1 protein. That is, the amino acid sequence represented by SEQ ID NO: 57 is included in the amino acid sequence of the Ror1 protein. Therefore, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57 encompasses the Ror1 protein. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57 can be obtained by purification from cells expressing the polypeptide using known methods. Alternatively, the polypeptide can be obtained by in vitro translation in a cell-free system. The cysteine-rich domain is present in the extracellular region of the Ror1 protein. As shown in the Examples below, the present inventors have discovered that the Ror1 protein can bind to at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein via the cysteine-rich domain. Therefore, a substance that has the effect of inhibiting the binding of a polypeptide containing the cysteine-rich domain to at least one Wnt protein selected from the group consisting of Wnt10a, Wnt16, and Wnt5a is highly likely to have the effect of inhibiting Ror1 signaling.

[0030] Examples of polypeptides comprising the amino acid sequence represented by SEQ ID NO:57 include Ror1 protein; polypeptides comprising the amino acid sequence represented by SEQ ID NO:57 and having a sequence identity of 85% or more (preferably 90% or more, more preferably 95% or more) to the amino acid sequence represented by SEQ ID NO:57; polypeptides comprising an amino acid sequence in which 1 to 50 amino acids (preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 to 5) have been added to the amino acid sequence represented by SEQ ID NO:57; and polypeptides comprising the amino acid sequence represented by SEQ ID NO:57. These polypeptides are also encompassed by the present disclosure as long as they are capable of interacting with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. Like Ror1 protein, polypeptides comprising the amino acid sequence represented by SEQ ID NO:57 may be bound to or fused with any other peptide, oligopeptide, or protein, as long as they are capable of interacting with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein.

[0031] (iv-a) Ror2 Protein-Expressing Cells Ror2 protein-expressing cells are not particularly limited as long as they express Ror2 protein. For example, they may be glioblastoma cells (e.g., T98G cells or U251MG cells) or cells in which Ror2 protein is forcibly expressed (e.g., HEK293 cells). Furthermore, Ror2 protein-expressing cells may be, for example, primary culture cells, passaged cells, or established cell lines.

[0032] (iv-b) Ror2 Protein In the screening method of the present disclosure, the Ror2 protein may be Ror2 protein expressed in Ror2 protein-expressing cells, such as glioblastoma cells, as is, or Ror2 protein purified by known methods from cells in which Ror2 protein is forcibly expressed. Alternatively, Ror2 protein obtained by in vitro translation in a cell-free system may be used. Specifically, the Ror2 protein of the present disclosure includes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 60, and a protein consisting of an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 60 and that can interact with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, and more preferably 95% or more.

[0033] Furthermore, the Ror2 protein of the present disclosure encompasses proteins consisting of an amino acid sequence in which 1 to 100 (preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5) amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence represented by SEQ ID NO: 60, and which can interact with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. The Ror2 protein may be bound to or fused with any other peptide, oligopeptide, or protein, as long as it can interact with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. Examples of other peptides, oligopeptides, or proteins include albumin (e.g., serum albumin), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag), fluorescent proteins (e.g., GFP, BFP, CFP, YFP, RFP), luminescent proteins (e.g., luciferase), secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression-enhancing sequences, solubilization sequences, and multimerization domains.

[0034] (iv-c) Polypeptide Comprising the Amino Acid Sequence Represented by SEQ ID NO: 58 The amino acid sequence represented by SEQ ID NO: 58 is the amino acid sequence constituting the cysteine-rich domain (sometimes referred to as "CRD" in the present disclosure) of the Ror2 protein. That is, the amino acid sequence represented by SEQ ID NO: 58 is included in the amino acid sequence of the Ror2 protein. Therefore, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58 encompasses the Ror2 protein. The polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58 can be obtained by purification from cells expressing the polypeptide using known methods. The polypeptide can also be obtained by in vitro translation in a cell-free system. The cysteine-rich domain is present in the extracellular region of the Ror2 protein. As shown in the Examples below, the present inventors have discovered that the Ror2 protein can bind to at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein via the cysteine-rich domain. Therefore, a substance that has the effect of inhibiting the binding of a polypeptide containing the cysteine-rich domain to at least one Wnt protein selected from the group consisting of Wnt10a, Wnt16, and Wnt5a is highly likely to have an inhibitory effect on Ror2 signaling.

[0035] Examples of polypeptides comprising the amino acid sequence represented by SEQ ID NO:58 include Ror2 protein; polypeptides comprising the amino acid sequence represented by SEQ ID NO:58 and having a sequence identity of 85% or more (preferably 90% or more, more preferably 95% or more) to the amino acid sequence represented by SEQ ID NO:58; polypeptides comprising an amino acid sequence in which 1 to 50 amino acids (preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 to 5) have been added to the amino acid sequence represented by SEQ ID NO:58; and polypeptides comprising the amino acid sequence represented by SEQ ID NO:58. These polypeptides are also encompassed by the present disclosure as long as they are capable of interacting with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein. Like Ror2 protein, polypeptides comprising the amino acid sequence represented by SEQ ID NO:58 may be bound to or fused with any other peptide, oligopeptide, or protein, as long as they are capable of interacting with at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein.

[0036] There are no particular limitations on the method for contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein; (ii) a test substance; and at least one selected from the group consisting of (iii-a) Ror1 protein-expressing cells, (iii-b) Ror1 protein, (iii-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57, (iv-a) Ror2 protein-expressing cells, (iv-b) Ror2 protein, and (iv-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58. For example, (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, and (ii) the test substance may be added to a culture medium for (iii-a) Ror1 protein-expressing cells and / or (iv-a) Ror2 protein-expressing cells. Alternatively, (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein; (ii) a test substance; and at least one selected from the group consisting of (iii-b) Ror1 protein, (iii-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57, (iv-b) Ror2 protein, and (iv-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58 may be mixed in any buffer.

[0037] The concentration (total concentration) of the Wnt protein during the contact is not particularly limited. For example, it can be about 0.1 to 4 μg / ml. Furthermore, the concentration of at least one selected from the group consisting of (iii-a) Ror1 protein-expressing cells, (iii-b) Ror1 protein, (iii-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57, (iv-a) Ror2 protein-expressing cells, (iv-b) Ror2 protein, and (iv-c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58 is also not particularly limited. The concentration of the test substance can be appropriately set depending on the type and properties of the test substance.

[0038] The temperature during contact is not particularly limited. For example, it can be about 25 to 40°C, or about 30 to 40°C. The contact time is not particularly limited. For example, it can be about 30 minutes to 60 hours, or about 1 hour to 48 hours. During contact, stirring, shaking, etc. can be performed as necessary.

[0039] The Pla2g2a gene encodes a phospholipase A2 group 2A protein. Pla2g2a is a secreted lipid metabolic enzyme. For example, the accession number for the mRNA sequence of the human Pla2g2a gene is NM_000300.

[0040] In step (A), the suppression of Pla2g2a gene expression in Ror1 protein-expressing cells can be confirmed by measuring the expression level of Pla2g2a mRNA or protein. Methods for measuring the expression level of mRNA include, for example, quantitative RT-PCR, in situ hybridization, and FISH. Methods for measuring the expression level of protein include, for example, Western blotting, ELISA, and immunostaining.

[0041] It can be confirmed by conventionally known methods or methods that can be easily conceived by a person skilled in the art from conventionally known methods that the test substance inhibits the binding of the Wnt protein and the Ror1 protein in step (B), that the test substance inhibits the binding of the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57 in step (C), that the test substance inhibits the binding of the Wnt protein and the Ror2 protein in step (E), and that the test substance inhibits the binding of the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58 in step (G). Specifically, these can be confirmed by, for example, co-immunoprecipitation, pull-down assay, ELISA, surface plasmon resonance, AlphaScreen, analysis using a Biacore system, etc.

[0042] In step (D), the suppression of Nrf2 activation in Ror2-expressing cells can be confirmed by measuring the amount of Nrf2 present in the cell nucleus. Examples of methods for measuring the amount of Nrf2 present in the cell nucleus include immunostaining.

[0043] The Nrf2 (NF-E2-related factor 2) protein is a transcriptional regulator with a basic leucine zipper structure, and is known to control the expression of genes involved in oxidative stress resistance, detoxification metabolism, and the like. Normally, the Nrf2 protein binds to the Keap1 protein, and the Nrf2 / Keap1 complex is rapidly degraded. On the other hand, when cells are exposed to stimuli such as oxidative stress, the Nrf2 protein dissociates from the Keap1 protein, becomes activated, and translocates into the nucleus. For example, the accession number for the mRNA sequence of the human Nrf2 gene is NM_006164.

[0044] The specific method for contacting the Ror2 protein-expressing cells with the ferroptosis inducer in step (F) is not particularly limited. For example, the ferroptosis inducer may be added to a culture medium for the Ror2 protein-expressing cells. Examples of ferroptosis inducers include erastin and RSL3. The concentration of the ferroptosis inducer can be appropriately set depending on the type and properties of the ferroptosis inducer, and may be, for example, about 1 to 10 μM. Furthermore, a washing step may be included before the contact. The washing method is not particularly limited, and a known method can be selected.

[0045] The temperature during contact is not particularly limited. For example, it can be about 25 to 40°C, or about 30 to 40°C. The contact time is not particularly limited. For example, it can be about 30 minutes to 60 hours, or about 1 hour to 48 hours. During contact, stirring, shaking, etc. can be performed as necessary.

[0046] Furthermore, the method for measuring the viability of Ror2 protein-expressing cells in step (F) is not particularly limited. For example, the viability of cells can be measured using WST-8 reagent.

[0047]

[0023] As used herein, "stemness of glioblastoma cells" refers to the stem cell-like, undifferentiated properties of glioblastoma cells. As shown in the Examples below, when the expression level of the Ror1 gene in glioblastoma cells increases, the expression level of the Pla2g2a gene increases, enhancing the stemness of glioblastoma cells; and, because the stemness of glioblastoma cells is suppressed by suppressing the expression of the Pla2g2a gene, it is speculated that Ror1 signaling controls the stemness of glioblastoma cells via the increased expression of the Pla2g2a gene.

[0048] In step (A) of the screening method of the present disclosure, if the expression of the Pla2g2a gene in Ror1 protein-expressing cells is suppressed when the test substance is used compared to when the test substance is not used, then the test substance can be said to have an inhibitory effect on the stemness of glioblastoma cells, and ultimately to have an inhibitory effect on the progression of glioblastoma cells.

[0049] In step (B) of the screening method of the present disclosure, if the binding of at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein to Ror1 protein is inhibited when the test substance is used compared to when the test substance is not used, the test substance can be said to have an inhibitory effect on the stemness of glioblastoma cells and, therefore, an inhibitory effect on the progression of glioblastoma cells. Similarly, in step (C) of the screening method of the present disclosure, if the binding of at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein to a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57 is inhibited when the test substance is used compared to when the test substance is not used, the test substance can be said to have an inhibitory effect on the stemness of glioblastoma cells and, therefore, an inhibitory effect on the progression of glioblastoma cells.

[0050] That is, at least one of steps (A) to (C) can be used to screen for substances that suppress the stemness of glioblastoma cells. It is presumed that suppressing the stemness of glioblastoma cells can suppress the progression of glioblastoma.

[0051] Many cancer cells, including glioblastoma, are known to overexpress the Slc7a11 gene, which encodes a cystine transporter, and to avoid excessive oxidative stress by promoting cystine uptake. Furthermore, Slc7a11 and ferritin, an iron storage protein that regulates intracellular iron metabolism, are known to contribute to the enhancement of tumorigenicity or drug resistance of cancer cells by suppressing ferroptosis (iron-dependent cell death).

[0052] As shown in the Examples below, in glioblastoma cells, suppression of Ror2 gene expression reduces the Nrf2 level in the cell nucleus, and reduces the expression of genes that suppress ferroptosis, such as the Slc7a11 gene and the Ftl (ferritin light chain) gene and Fth1 (ferritin heavy chain) gene, which encode ferritin. Therefore, it is speculated that the Ror2 signal promotes the expression of ferroptosis resistance genes via activation of Nrf2, thereby contributing to the enhancement of ferroptosis resistance in glioblastoma cells.

[0053] In step (D) of the screening method of the present disclosure, if the activation of Nrf2 protein in Ror2 protein-expressing cells is suppressed when the test substance is used compared to when the test substance is not used, the test substance can be said to have an effect of suppressing the ferroptosis resistance of glioblastoma cells, and ultimately to have an effect of suppressing the progression of glioblastoma cells. It is presumed that the suppression of ferroptosis resistance of glioblastoma cells can suppress the progression of glioblastoma.

[0054] In step (E) of the screening method of the present disclosure, if the binding of Wnt protein and Ror2 protein is inhibited when the test substance is used compared to when the test substance is not used, the test substance can be said to have an effect of suppressing ferroptosis resistance in glioblastoma cells, and therefore to have an effect of suppressing the progression of glioblastoma cells. Similarly, in step (G) of the screening method of the present disclosure, if the binding of Wnt protein and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 58 is inhibited when the test substance is used compared to when the test substance is not used, the test substance can be said to have an effect of suppressing ferroptosis resistance in glioblastoma cells, and therefore to have an effect of suppressing the progression of glioblastoma cells.

[0055] In step (F) of the screening method of the present disclosure, if the survival rate of Ror2 protein-expressing cells is lower when the test substance is used compared to when the test substance is not used, it can be said that the test substance has an effect of suppressing the ferroptosis resistance of glioblastoma cells, and therefore has an effect of suppressing the progression of glioblastoma cells.

[0056] That is, by performing at least one of steps (D) to (G), it is possible to screen for a substance that suppresses the ferroptosis resistance of glioblastoma cells. It is presumed that suppressing the ferroptosis resistance of glioblastoma cells can suppress the progression of glioblastoma.

[0057] Although Ror1 and Ror2 play important roles in tissue or organ formation during development and in the progression of cancer, it has been reported that their expression is barely observed in normal adult tissues. Therefore, targeting Ror1 and / or Ror2 as a therapeutic target for glioblastoma is expected to lead to the development of highly effective treatments with fewer side effects, which is considered to be one of the major advantages of this discovery.

[0058] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0059] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0060] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these examples. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "volume %."

[0061] Adherent culture: Human glioblastoma cell lines, T98G cells and U251MG cells, were suspended in DMEM medium containing 10% (v / v) fetal bovine serum (FBS), seeded on a culture dish, and incubated at 37°C and 5% CO ２ In some experiments, the medium was replaced with serum-free medium (Opti-MEM, Thermo Fisher Scientific, Waltham, MA) the day after culturing, and 24 hours later, cells were stimulated with recombinant PLA2G2A protein (in PBS containing 0.1% (w / v) BSA, final concentration 200 ng / ml, R&D Systems, Minneapolis, MN) or solvent alone (PBS containing 0.1% (w / v) BSA).

[0062] In knockdown experiments, various siRNAs (final concentration 10 nM each) were introduced into cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific). The target sequences of the siRNAs are listed in Table 1. In forced expression experiments of the Ror1 gene, a Ror1 gene expression plasmid vector (pcDNA Ror1) or an empty vector (pcDNA) was introduced into T98G cells by electroporation together with a plasmid vector expressing a puromycin resistance gene. The cells were then cultured for 2 days in a medium containing puromycin (final concentration 2 μg / ml, Nacalai Tesche, Tokyo, Japan) to perform drug selection of the transiently gene-transfected cells. In the forced expression experiment of the Nrf2 gene, U251MG cells were infected with a retroviral vector expressing Nrf2 fused with the ligand-binding domain of the estrogen receptor together with a puromycin resistance gene (ERT2-Nrf2) or an empty retroviral vector (mock), and then cultured in a medium containing 2 μg / ml puromycin for 2 weeks to perform drug selection of stable-expressing cell lines.

[0063]

[0064] Spheroid culture: T98G and U251MG cells that had been subjected to adhesion culture were detached from the dish using Accutase (Nacalai Tesche) and suspended in DMEM / F12 medium containing MACS Neurobrew-21 (Miltenyi Biotec, Tokyo, Japan), bFGF (final concentration 20 μg / ml, PeproTech, Cranbury, NJ), and EGF (final concentration 20 μg / ml EGF, FUJIFILM Wako Pure Chemical, Tokyo, Japan). The cells were then seeded onto a Petri dish and incubated at 37°C and 5% CO. ２ For Ror1 function inhibition experiments, strictinin (final concentration 50 μM, Nagara Science, Gifu, Japan) or its solvent (DMSO) was added to the medium.

[0065] Quantitative RT-PCR (Quantitative Reverse Transcription-PCR) Cultured cells were solubilized using Sepasol RNA I SuperG (Nacalai Tesche) and total RNA was collected. cDNA was synthesized from the collected total RNA using PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan). Real-time PCR was performed using the synthesized cDNA on a LightCycler 480 II system (Roche, Basel, Switzerland) to quantitatively analyze the expression levels of the target genes. The expression levels of each gene were normalized using the expression level of the B2M gene as an internal standard. The sequences of the primer pairs are listed in Table 2.

[0066]

[0067] cDNAs encoding recombinant proteins (Ror1-Fc and Ror2-Fc) in which the extracellular regions of human Ror1 and Ror2 were fused with the Fc domain of human IgG, respectively, and mutant Ror1-Fc (Ror1ΔCRD-Fc) and Ror2-Fc (Ror2ΔCRD-Fc) lacking the cysteine-rich domain involved in binding to Wnt5a, were inserted into the pcDNA5 FRT vector (Thermo Fisher Scientific). The sequences of the cysteine-rich domains are listed in Table 3. Using these vectors, CHO cell lines stably expressing Ror1-Fc, Ror2-Fc, Ror1ΔCRD-Fc, and Ror2ΔCRD-Fc were generated using the Flp-In System (Thermo Fisher Scientific). The generated cell lines were cultured in 10% (v / v) FBS-containing DMEM / F12 medium until confluent. The medium was then replaced with serum-free DMEM / F12 medium, and the culture supernatant was collected after 48 hours. Various recombinant Ror-Fc proteins were purified using a Protein A Sepharose column.

[0068]

[0069] Binding analysis of Ror-Fc with Wnt proteins. Human IgG Fc (R&D Systems, Minneapolis, MN), and recombinant proteins of Ror1-Fc and Ror2-Fc were each bound to Protein A / G magnetic beads (Thermo Fisher Scientific) and then incubated with purified Wnt5a protein or purified Wnt16 protein. The amount of Wnt5a or Wnt16 bound to each magnetic bead was analyzed by Western blotting. The primary antibodies used were anti-Wnt5a antibody (Cell Signaling Technology), anti-Wnt16 antibody (Proteintech, Tokyo, Japan), and anti-IgG-Fc antibody (Tokyo Chemical Industry, Tokyo, Japan). For secondary antibody and signal detection, goat anti-rabbit IgG (H+L)-HRP conjugate, goat anti-mouse IgG (H+L)-HRP conjugate (Bio-Rad Laboratories, Tokyo, Japan), and SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) were used. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. After each step, the sections were washed three times for 10 minutes with 1x PBS-T. Furthermore, mutant Ror1-Fc (Ror1ΔCRD-Fc) and mutant Ror2-Fc (Ror2ΔCRD-Fc) lacking the cysteine-rich domain (CRD) were also analyzed in the same manner.

[0070] Establishment of stable cell lines To establish a T98G cell line expressing the soluble extracellular domain of Ror1 (Ror1 decoy), which is thought to function as a decoy receptor for Ror1, T98G cells were infected with a retroviral vector expressing the extracellular domain of Ror1 together with a puromycin resistance gene (Ror1 decoy), or an empty retroviral vector (mock) as a control. Subsequently, drug selection of stable expression cell lines was performed by culturing in a medium containing 2 μg / ml puromycin for 2 weeks. Furthermore, to establish a Wnt10a-expressing Cos7 cell line, Cos7 cells were infected with a lentiviral vector expressing human Wnt10a together with a blasticidin resistance gene, or an empty lentiviral vector (mock) as a control. Thereafter, the cells were cultured for 2 weeks in a medium containing 10 μg / ml blasticidin S (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to perform drug selection of the stably expressing cell line.

[0071] Preparation of Wnt10a-containing conditioned medium: Wnt10a-expressing Cos7 cells or empty vector-transfected Cos7 cells prepared by the method described above were cultured in 10% (v / v) FBS-containing DMEM medium until confluent. The medium was then replaced with serum-free DMEM / F12 medium. After 48 hours, the culture supernatant was collected and concentrated approximately 30-fold using an Amicon Ultra 10K (Merck Millipore, Billerica, MA). In the Wnt10a stimulation experiment, concentrated Wnt10a-containing culture supernatant (Wnt10a CM) or control culture supernatant (Mock CM) was added in an amount of 1 / 10 of the culture medium.

[0072] U251MG cells suspended in 10% (v / v) FBS-containing DMEM were seeded onto a 96-well plate and transfected with siRNA. After culturing for a given period, WST-8 reagent (Cell Counting Kit-8, Dojindo, Kumamoto, Japan) was added to the medium, followed by culturing for 90 minutes and measuring the absorbance at 450 nm.

[0073] Analysis of spheroid growth rate: T98G cells suspended in DMEM / F12 medium containing MACS Neurobrew-21, bFGF (final concentration 20 μg / ml), and EGF (final concentration 20 μg / ml) were seeded into ultra-low attachment surface-treated round-bottom 96-well plates (Corning, New York, NY) to form spheroids, which were then cultured for a desired period. Spheroid growth rates were then measured using the CellTiter-Glo 3D Cell Viability Assay (Promega, Madison, WI).

[0074] Analysis of Ferroptosis Resistance The medium of U251MG cells transfected with siRNA by the above-described method was replaced with a medium containing or not containing Erastin (Cayman Chemical, Ann Arbor, MI), a ferroptosis inducer, and the cells were cultured for 24 hours. In another experiment, U251MG cells cultured in DMEM / F12 medium containing MACS Neurobrew-21, bFGF (final concentration 20 μg / ml), and EGF (final concentration 20 μg / ml) were stimulated with Wnt5a, Wnt16, or Wnt10a-CM, and then cultured for 24 hours in medium containing or not containing the ferroptosis inducer RSL3 (Selleck Chemicals, Houston, TX). The number of viable cells was measured using WST-8 reagent, and the ratio of the number of viable cells in the medium containing erastin or RSL3 to the number of viable cells in the medium without the ferroptosis inducer was calculated as the relative viability. In the analysis using ERT2-Nrf2-expressing cells or mock cells, 4-hydroxytamoxifen (OHT, final concentration 300 nM, Sigma-Aldrich, St. Louis, MO) was added to the medium.

[0075] Immunostaining: U251MG cells cultured on glass coverslips were fixed with 4% (w / v) paraformaldehyde (PFA) / PBS for 20 minutes, treated with 0.2% (v / v) Triton X-100 / PBS for 20 minutes, and then blocked with 3% (w / v) BSA / PBS. Anti-Nrf2 antibody (Cell Signaling Technology, Danvers, MA) diluted with 3% (w / v) BSA / PBS was used as the primary antibody. The secondary antibody and signal detection were performed using the SuperBoost Tyramide Signal Kit (Thermo Fisher Scientific). The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated at room temperature for 1 hour. After each step, the cells were washed three times with 1x PBS for 10 minutes each. After immunostaining, the cell nuclei were stained with DAPI.

[0076] Bioinformatics analysis: Using cBioPortal, a cancer genome database analysis tool, data on glioma patients (grades 2-4, approximately 600 patients) registered in TCGA (The Cancer Genome Atlas) was obtained, and the expression levels of the Ror2 gene, Wnt10a gene, and Wnt16 gene were analyzed for each glioma grade. Survival curves were analyzed by classifying patients into two groups: those with the highest 50% expression level of each gene and those with the lowest 50% expression level.

[0077] Statistical analysis: Data were analyzed using GraphPad Prism 9.0 (GraphPad Software, Boston, MA). Data are presented as mean ± standard deviation (S.D.). Significance was determined using an unpaired two-tailed Student's t test when comparing two groups, or a one-way ANOVA with Dunnett's test when analyzing three or more groups, with *P<0.05, **P<0.01, or ***P<0.001.

[0078] The expression of various genes in T98G cells (top panel) and U251MG cells (bottom panel) cultured in either adherent or suspension culture was analyzed by quantitative RT-PCR. The results are shown in Figure 1. Compared to adherent culture, glioblastoma cells cultured in suspension to form spheroids showed increased expression of the Ror1 gene, Nanog gene and CD36 gene, which are marker genes for glioblastoma cell stemness, as well as the Pla2g2a gene.

[0079] T98G cells that formed spheroids in suspension culture were treated with strictinin (final concentration 50 μM), a Ror1 inhibitor, and the effect on various gene expression was analyzed by quantitative RT-PCR. The results are shown in Figure 2. It was shown that strictinin treatment reduced the expression of Pla2g2a gene and stemness marker genes (Nanog gene and CD36 gene).

[0080] The effect of forced expression of Ror1 on the expression of various genes in adherently cultured T98G cells was analyzed by quantitative RT-PCR. The results are shown in Figure 3. Forced expression of Ror1 was shown to increase the expression of Pla2g2a gene and stemness marker genes (Nanog gene and CD36 gene).

[0081] The effect of knocking down Pla2g2a gene expression using Pla2g2a siRNA in T98G cells that formed spheroids in suspension culture on the expression of various genes was analyzed by quantitative RT-PCR. The results are shown in Figure 4. It was shown that suppression of Pla2g2a gene expression reduced the expression of stemness marker genes (Nanog gene and CD36 gene).

[0082] The effect of stimulation of adherently cultured T98G cells with recombinant PLA2G2A protein on the expression of various genes was analyzed by quantitative RT-PCR. The results are shown in Figure 5. It was shown that PLA2G2A stimulation increased the expression of stemness marker genes (Nanog gene and CD36 gene).

[0083] These findings suggest that Ror1 signaling may regulate the stemness of glioblastoma cells through increased expression of Pla2g2a. Nanog and CD36 are marker genes expressed in undifferentiated stem cells, and their expression is presumed to increase due to enhanced stemness via the function of Pla2g2a.

[0084] As shown in Figure 6, bioinformatics analysis using the TCAG database demonstrated that Ror2 was highly expressed in tumor tissues from glioblastoma patients (grade 4). High expression of Ror1 in glioblastoma has also been reported (Non-Patent Document 2). Furthermore, survival curve analysis demonstrated that the higher the expression levels of Ror1 and Ror2 in glioblastoma, the worse the prognosis.

[0085] The expression of the Ror2 gene in T98G and U251MG cells cultured in adherent or suspension culture was analyzed by quantitative RT-PCR. The results are shown in Figure 7. We found that Ror2 gene expression was elevated in glioblastoma cells that formed spheroids in suspension culture compared to adherent culture.

[0086] The relative cell proliferation ability and the relative amount of Nrf2 protein localized in the nucleus were analyzed when Ror2 gene expression in U251MG cells was knocked down using siRNA. The results are shown in Figure 8. It was shown that inhibition of Ror2 expression reduced proliferation ability and the level of Nrf2 in the nucleus.

[0087] The effect of siRNA-mediated knockdown of the Nrf2 or Ror2 gene in U251MG cells on the expression of Nrf2 target genes was analyzed by quantitative RT-PCR. The results are shown in Figure 9. We found that suppression of Nrf2 or Ror2 gene expression reduced the expression of known Nrf2 target genes, including the Slc7a11 gene, which encodes a cystine transporter, as well as the Ftl (ferritin light chain) and Fth1 (ferritin heavy chain) genes, which encode ferritin, an iron storage protein responsible for regulating intracellular iron metabolism.

[0088] We analyzed the effect of suppressing Ror2 gene expression in U251MG cells on cell death induced by erastin (a ferroptosis inducer). The results are shown in Figure 10. It was demonstrated that suppressing Ror2 gene expression increased erastin sensitivity. Furthermore, using U251MG cells overexpressing Nrf2 fused to the ligand-binding domain of the estrogen receptor (ERT2-Nrf2), treatment with 4-hydroxytamoxifen (OHT) significantly induced Slc7a11 gene expression by translocating ERT2-Nrf2 to the nucleus. It was also demonstrated that suppressing Ror2 gene expression in U251MG cells with OHT treatment did not result in a decrease in Slc7a11 gene expression or an increase in erastin sensitivity. These findings suggest that Ror2 signaling contributes to the acquisition of ferroptosis resistance in glioblastoma cells via activation of Nrf2.

[0089] As shown in Figure 11, bioinformatics analysis using the TCAG database demonstrated high expression of Wnt10a and Wnt16 in tumor tissues from glioblastoma patients (grade 4). High expression of Wnt5a in glioblastoma has also been reported (Non-Patent Document 2). Furthermore, survival curve analysis demonstrated that higher expression levels of Wnt10a and Wnt16 in glioblastoma correlated with poorer prognosis.

[0090] The expression of the Wnt5a gene, Wnt10a gene, and Wnt16 gene in T98G and U251MG cells cultured in either adherent or suspension culture was analyzed by quantitative RT-PCR. The results are shown in Figure 12. We found that expression of the Wnt10a gene and Wnt16 gene increased in glioblastoma cells that formed spheroids in suspension culture compared to adherent culture. These findings suggest that Wnt10a or Wnt16 is involved in the activation of Ror1 and Ror2 signaling in glioblastoma cells (spheroid-forming glioblastoma cells).

[0091] The expression of Ror1 in T98G cells that formed spheroids in suspension culture was knocked down using two types of Ror1 siRNA (#1 and #2) with different sequences, and the effects on the expression of various genes were analyzed by quantitative RT-PCR. The results are shown in Figure 13. As shown in Figure 13, suppression of Ror1 expression reduced the expression of Pla2g2a.

[0092] The expression of Wnt5a in T98G cells that formed spheroids in suspension culture was knocked down using two types of Wnt5a siRNA (#1 and #2) with different sequences, and the effects on the expression of various genes were analyzed by quantitative RT-PCR. The results are shown in Figure 14. As shown in Figure 14, suppression of Wnt5a expression reduced the expression of Pla2g2a.

[0093] We prepared recombinant proteins, Ror1-Fc and Ror2-Fc, by fusing the extracellular domain of Ror1 and Ror2 with the Fc domain of IgG, respectively. The purified Ror1-Fc and Ror2-Fc proteins were bound to Protein A / G magnetic beads and then incubated with purified Wnt5a or Wnt16 protein. Magnetic beads bound only to the Fc domain of IgG (Fc) were used as a control. The amounts of Wnt5a and Wnt16 bound to each magnetic bead were then analyzed by Western blotting. The results are shown in Figure 15. As shown in Figure 15, both Ror1-Fc and Ror2-Fc bound to Wnt5a and Wnt16. These results suggest that Ror1 and Ror2 function as receptors for Wnt16 in addition to Wnt5a.

[0094] We prepared mutant Ror1-Fc (Ror1ΔCRD-Fc) and mutant Ror2-Fc (Ror2ΔCRD-Fc) lacking the cysteine-rich domain (CRD) involved in binding to Wnt5a. Various Ror-Fc proteins were bound to Protein A / G magnetic beads and then incubated with purified Wnt16 protein. The amount of Wnt16 bound to each magnetic bead was then analyzed using Western blotting. The results are shown in Figure 16. As shown in Figure 16, deletion of the CRD from Ror1 and Ror2 reduced the amount of Wnt16 binding. More specifically, as shown in the lower panel of Figure 16, although the amount of protein subjected to Western blotting was greater for Ror1ΔCRD-Fc and Ror2ΔCRD-Fc than for Ror1-Fc and Ror2-Fc, the amount of bound Wnt16 was less for Ror1ΔCRD-Fc and Ror2ΔCRD-Fc than for Ror1-Fc and Ror2-Fc, as shown in the upper panel of Figure 16. These results suggest that Ror1 and Ror2 bind to Wnt16 via their respective CRDs.

[0095] In T98G cells that formed spheroids, the effect of forced expression of the extracellular domain of Ror1 as Ror1 decoy on the expression level of Pla2g2a and stemness was analyzed. The results are shown in Figure 17. As shown in Figure 17, analysis using quantitative RT-PCR demonstrated that forced expression of Ror1 decoy reduced Pla2g2a expression. Furthermore, it was shown that forced expression of Ror1 decoy reduced the growth rate of spheroids.

[0096] We analyzed the effect on spheroid growth rate of spheroid-forming T98G cells stimulated with purified Wnt16 protein. The results are shown in Figure 18. As shown in Figure 18, Wnt16 stimulation increased the spheroid growth rate. Furthermore, forced expression of Ror1 decoy suppressed the increase in spheroid growth rate due to Wnt16 stimulation.

[0097] The effect of stimulating U251MG cells with purified Wnt5a protein or purified Wnt16 protein on cell death induced by RSL3 (a ferroptosis inducer) was analyzed. Similarly, the effect of stimulating U251MG cells with culture supernatant (Wnt10a CM) of Wnt10a-expressing Cos7 cells on cell death induced by RSL3 was analyzed. The results are shown in Figure 19. As shown on the left side of Figure 19, cells stimulated with Wnt5a or Wnt16 showed enhanced resistance to ferroptosis compared to cells treated with solvent (PBS) alone. Furthermore, as shown on the right side of Figure 19, cells treated with Wnt10a CM showed enhanced resistance to ferroptosis compared to cells treated with culture supernatant (mock CM) of Cos7 cells expressing an empty vector.

[0098] From these test results, we hypothesized a Ror1 signaling pathway initiated by the binding of at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein to Ror1 protein. We also hypothesized a Ror2 signaling pathway initiated by the binding of at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein to Ror2 protein (Figure 20, left). Furthermore, from these test results, we discovered a screening strategy for substances that have an inhibitory effect on glioblastoma cell progression, as shown in Figure 20, right.

Claims

1. A method for screening for a substance having an inhibitory effect on the progression of glioblastoma cells, comprising the steps of: evaluating the Ror1 signaling inhibitory effect of a test substance; and / or evaluating the Ror2 signaling inhibitory effect of the test substance, wherein the Ror1 signaling inhibitory effect is confirmed by: (A) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-a) a Ror1 protein-expressing cell, and then confirming that expression of Pla2g2a gene is suppressed in the Ror1 protein-expressing cell; (B) contacting (i) at least one Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iii-a) a Ror1 protein, and then (C) (i) a step of confirming that the test substance inhibits the binding of a Wnt protein and a Ror1 protein; and (C) a step of confirming that the test substance inhibits the binding of a Wnt protein and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57 after contacting (i) at least one Wnt protein selected from the group consisting of a Wnt10a protein, a Wnt16 protein, and a Wnt5a protein, (ii) the test substance, and (iii-b) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57; and the Ror2 signaling inhibitory action is evaluated by a method comprising at least one step selected from the group consisting of: (D) (i) a step of confirming that activation of Nrf2 protein in a Ror2 protein-expressing cell is suppressed after contacting (i) at least one Wnt protein selected from the group consisting of a Wnt10a protein, a Wnt16 protein, and a Wnt5a protein, (ii) the test substance, and (iv-a) a Ror2 protein-expressing cell;(E) (i) at least one kind of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-a) Ror2 protein, and then confirming that the test substance inhibits binding between the Wnt protein and the Ror2 protein; (F) (i) at least one kind of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-a) Ror2 protein-expressing cells, and then contacting the Ror2 protein-expressing cells with a ferroptosis inducer, and then measuring the viability of the Ror2 protein-expressing cells; and (G) (i) at least one kind of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) the test substance, and (iv-b) a step of contacting a test substance with a polypeptide comprising the amino acid sequence represented by SEQ ID NO:58, and then confirming that the test substance inhibits binding between a Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO:

58.

2. The screening method according to claim 1, comprising: a step of evaluating the Ror1 signaling inhibitory effect of a test substance; and a step of evaluating the Ror2 signaling inhibitory effect of the test substance.

3. The screening method according to claim 1 or 2, wherein the Ror1 signal transduction inhibitory effect is evaluated by a method comprising the steps of: (A) contacting (i) at least one type of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iii-a) Ror1 protein-expressing cells, and then confirming that expression of Pla2g2a gene is suppressed in Ror1 protein-expressing cells.

4. The screening method according to claim 1 or 2, wherein the Ror2 signal transduction inhibitory effect is evaluated by a method comprising: (D) a step of contacting (i) at least one type of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iv-a) a Ror2 protein-expressing cell, and then confirming that activation of Nrf2 protein in the Ror2 protein-expressing cell is suppressed.

5. The screening method according to claim 1 or 2, wherein the Ror1 signaling inhibitory effect is evaluated by a method comprising the steps of: (C) contacting (i) at least one type of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iii-b) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:57, and then confirming that the test substance inhibits binding between the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO:

57.

6. The screening method according to claim 1 or 2, wherein the Ror2 signaling inhibitory effect is evaluated by a method comprising the steps of: (G) contacting (i) at least one type of Wnt protein selected from the group consisting of Wnt10a protein, Wnt16 protein, and Wnt5a protein, (ii) a test substance, and (iv-b) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:58, and then confirming that the test substance inhibits binding between the Wnt protein and the polypeptide comprising the amino acid sequence represented by SEQ ID NO:58.

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