WO2018088426A1 - Anticancer agent and use thereof - Google Patents

Abstract

Provided is an anticancer agent that contains an inhibitor of StAR-related lipid transfer domain containing 3 (STARD3)/cholesterol change/C-Met pathway as an active ingredient.

Description

Anticancer agent and use thereof

The present invention relates to an anticancer agent and use thereof. More specifically, the present invention relates to an anticancer agent, a pharmaceutical composition for cancer treatment, and a screening method for an anticancer agent. This application claims priority based on Japanese Patent Application No. 2016-217890 filed in Japan on November 8, 2016, the contents of which are incorporated herein by reference.

Cancer is one of the leading causes of death in modern society. Many efforts have been made to develop cancer therapies, but they are still often difficult to cure, with the exception of early cancers that can be surgically removed. In particular, melanoma (malignant melanoma), triple negative breast cancer, and liver cancer have a poor prognosis, and the development of a treatment method thereof is demanded.

多 く Many inoperable cancers are treated with chemotherapy or radiation therapy. However, it is extremely difficult to completely eliminate tumor cells, and in many cases, treatment-resistant cells appear and recur.

Recently, the cancer stem cell hypothesis has been proposed as a cause of cancer recurrence and metastasis (see, for example, Non-Patent Document 1). According to the cancer stem cell hypothesis, there are stem cells similar to normal tissues in tumor tissue, and they have the ability to replicate themselves, and the ability to form tumors similar to the original tumor tissue with only a few. It is thought to have. And since cancer stem cells have resistance to anticancer drugs and radiation, they are likely to remain during treatment and are considered to cause recurrence and metastasis.

Under such circumstances, establishment of a cancer treatment method that is effective for cancer stem cells and has a low risk of recurrence and metastasis is demanded. Then, an object of this invention is to provide a new anticancer agent.

The present invention includes the following aspects.
[1] An anticancer agent comprising, as an active ingredient, an inhibitor of StAR-related lipid transfer domain containing 3 (STARD3) / cholesterol change / c-Met pathway.
[2] The anticancer agent according to [1], wherein the STARD3 / cholesterol change / c-Met pathway inhibitor is a STARD3 inhibitor, a cholesterol kinetic regulator, or a c-Met inhibitor.
[3] The anticancer agent according to [1] or [2], wherein the inhibitor of STARD3 / cholesterol change / c-Met pathway is a 67 kDa laminin receptor (67LR) agonist.
[4] The anticancer agent according to any one of [1] to [3], wherein the 67LR agonist is epigallocatechin gallate or a derivative thereof.
[5] The anticancer agent according to any one of [1] to [4], wherein the cancer is melanoma, breast cancer or liver cancer.
[6] The anticancer agent according to [5], wherein the breast cancer is triple negative breast cancer.
[7] The anticancer agent according to any one of [1] to [6], which is a cancer stem cell inhibitor.
[8] The anticancer agent according to any one of [1] to [7], which is a cancer metastasis inhibitor.
[9] A pharmaceutical composition for cancer treatment comprising the anticancer agent according to any one of [1] to [8] and a pharmaceutically acceptable carrier.
[10] The step of measuring the expression level of STARD3 in the cell in the presence of the test substance, and when the expression level of STARD3 is lower than the expression level in the absence of the test substance, And a step of determining that the test substance is an anticancer agent.
[11] The step of measuring the activity of STARD3 in the presence of the test substance, and the test substance is an anticancer agent when the activity of STARD3 is lower than the activity in the absence of the test substance A method for screening an anticancer agent.
[12] A step of measuring the abundance of cholesterol in a cell in the presence of a test substance, and when the abundance of cholesterol is reduced compared to the abundance in the absence of the test substance, And a step of determining that the test substance is an anticancer agent.
[13] The step of measuring the expression level of c-Met in cells in the presence of the test substance, and the expression level of c-Met was lower than the expression level in the absence of the test substance A method for screening an anticancer agent, comprising the step of determining that the test substance is an anticancer agent.
[14] A step of measuring the activity of c-Met in the presence of the test substance, and the test substance when the activity of c-Met is lower than that in the absence of the test substance And a step of judging that it is an anticancer agent.
[15] The method for screening an anticancer agent according to any one of [10] to [14], wherein the cancer is melanoma, breast cancer or liver cancer.
[16] The screening method for an anticancer agent according to [15], wherein the breast cancer is triple negative breast cancer.

According to the present invention, a new anticancer agent can be provided.

In Experimental example 1, it is a figure explaining the process which identified the STARD3 gene by the Genetic suppressor elements (GSE) method. (A) is a photograph showing the results of Western blotting in Experimental Example 2. (B) is the graph which digitized the result of (a). (A) and (b) are photomicrographs of control B16 cells (Scr-shRNA) and B16 cells (STARD3-shRNA) in which the expression of STARD3 was suppressed in Experimental Example 3, respectively. (C) is a graph showing the results of quantitative PCR in Experimental Example 3. (A) And (b) is a photograph which shows the result of the Western blot in Experimental example 4. FIG. (A) And (c) is a typical microscope picture which shows the result of the spheroid assay in Experimental example 5. FIG. (B) And (d) is a graph which shows the result of the spheroid assay in Experimental example 5. FIG. (A) is a representative fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 6. (B) is a graph summarizing the results of immunohistochemical staining of Experimental Example 6. 10 is a graph showing the results of examination in Experimental Example 7. (A) to (c) are graphs showing cell growth curves measured in Experimental Example 8. (A) is a representative photograph of a mouse (STARD3-shRNA) and a control mouse (Scr-shRNA) transplanted with B16 cells with suppressed expression of STARD3 in Experimental Example 9. (B) is a representative photograph showing a tumor tissue excised from each mouse in Experimental Example 9. (C) is a graph showing the measurement results of the tumor volume of each mouse in Experimental Example 9. (D) is a graph showing the survival curve of each mouse in Experimental Example 9. In Experimental example 10, it is the photograph of the lung extracted from each mouse | mouth. 10 is a graph showing the examination results of Experimental Example 11. (A) And (b) is a graph which shows the measurement result in Experimental example 12. FIG. (A)-(c) is a graph which shows the result of the spheroid assay in Experimental Example 13. (A) And (c) is a photograph which shows the result of the Western blot in Experimental example 14. FIG. (B) is the graph which digitized the result of (a). (D) is the graph which digitized the result of (c). 16 is a graph showing the results of a spheroid assay in Experimental Example 15. 16 is a graph showing the results of a spheroid assay in Experimental Example 16. 18 is a graph showing the results of Experimental Example 17. (A) is a photograph showing the results of Western blotting in Experimental Example 18. (B) is the graph which digitized the result of (a). (A) is a photograph showing the results of Western blotting in Experimental Example 19. (B) is the graph which digitized the result of (a). (A) is a photograph showing the results of Western blotting in Experimental Example 20. (B) is the graph which digitized the result of (a). (A) is a photograph showing the results of Western blotting in Experimental Example 21. FIG. (B) is the graph which digitized the result of (a). (C) is a graph showing the results of the spheroid assay in Experimental Example 21. 14 is a fluorescence micrograph showing the results of immunohistochemical staining in Experimental Example 22. (A) is a photograph showing the results of Western blotting in Experimental Example 23. FIG. (B) is the graph which digitized the result of (a). It is a graph which shows the result of the spheroid assay in Experimental example 24. (A) is the typical photograph of the lung extracted from the mouse in Experimental Example 25. (B) is a graph showing the measurement results of the number of lung metastasis nodules in Experimental Example 25. (C) is a graph showing the measurement results of lung weight in Experimental Example 25. (D) is a graph showing measurement results of AST activity in Experimental Example 25. (E) is a graph showing the measurement results of ALT activity in Experimental Example 25. It is a graph which shows the result of quantitative PCR in Experimental example 26. It is a photograph which shows the result of the Western blot in Experimental example 27. It is a graph which shows the result of the spheroid assay in Experimental example 28. FIG. 2 is a schematic diagram of STARD3 / change in cholesterol / c-Met pathway revealed by the inventors. (A) to (c) are photographs showing the results of Western blotting in Experimental Example 29 and graphs obtained by quantifying the results. (A) to (c) are graphs showing the results of the spheroid assay in Experimental Example 30. (A) And (b) is the photograph which showed the result of the western blot in Experimental example 31, and the graph which digitized the result. It is a graph which shows the result of the spheroid assay in Experimental example 32. (A) is a photograph showing the results of Western blotting in Experimental Example 33. FIG. (B) And (c) is the graph which digitized the result of (a). 16 is a graph showing the results of a spheroid assay in Experimental Example 34. (A) is a photograph showing the results of Western blotting in Experimental Example 35. FIG. (B) And (c) is the graph which digitized the result of (a).

[Anticancer agent]
In one embodiment, the present invention provides an anticancer agent comprising an inhibitor of STARD3 / cholesterol change / c-Met pathway as an active ingredient.

As described later in the Examples, the inventors have clarified a new mechanism by which cancer stem cells maintain cancer stem cell properties. FIG. 29 is a schematic diagram of STARD3 / change in cholesterol / c-Met pathway, which is a cancer stem cell maintenance pathway revealed by the inventors.

As shown in FIG. 29, when the expression of STARD3 increases, a change occurs in cholesterol as compared with the case where STARD3 is not expressed. Here, changes in cholesterol include changes in cholesterol kinetics such as changes in intracellular localization of cholesterol and changes in the amount of intracellular cholesterol. As cholesterol changes, c-Met expression increases and c-Met is activated. As a result, the cancer stem cell nature of the cells is maintained.

Here, STARD3 is a kind of membrane protein involved in cholesterol transport. RefSeq ID of human STARD3 mRNA is NM_001165937, NM_001165938, NM_006804, etc., and RefSeq ID of mouse STARD3 mRNA is NP_067522, NM_021547, etc.

C-Met is a receptor for hepatocyte growth factor (HGF). The RefSeq ID of human c-Met mRNA is NM_000245, NM_001127500, NM_001324401, and the RefSeq ID of mouse c-Met mRNA is NM_008591.

As described later in the Examples, growth of cancer cells can be suppressed by inhibiting the STARD3 / cholesterol change / c-Met pathway. Moreover, the function of cancer stem cells can be inhibited.

More specifically, the expression level of c-Met decreases when STARD3 activity in cancer cells is inhibited or when a cholesterol kinetic regulator is allowed to act on cancer cells. As a result, the expression level of Oct-4, CD271, JARID1B, etc., which are cancer stem cell markers, is decreased, the proliferation of cancer cells is suppressed, and the function of cancer stem cells is also inhibited.

Therefore, the anticancer agent of this embodiment can also be called a cancer stem cell inhibitor, a cancer metastasis inhibitor, or the like.

The cancer targeted by the anticancer agent of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer, or the like. In addition, the breast cancer to be treated by the anticancer agent of the present embodiment may be triple negative breast cancer. Triple negative breast cancer is breast cancer in which estrogen receptor (ER), progesterone receptor (PgR), and HER2 are all negative.

Melanoma, triple negative breast cancer, and liver cancer have poor prognosis, and effective therapeutic agents are required. On the other hand, as will be described later in Examples, the anticancer agent of this embodiment can effectively treat these cancers and can reduce the risk of recurrence and metastasis. Moreover, as will be described later in Examples, the anticancer agent of the present embodiment can suppress the functions of melanoma stem cells, breast cancer stem cells, and liver cancer stem cells. The breast cancer stem cell may be a cancer stem cell of triple negative breast cancer.

In the present specification, the cancer stem cell inhibitor means a substance that inhibits the function of cancer stem cells. The function of cancer stem cells can be said to be a function of forming a tumor similar to the original tumor tissue with a small number. The function of cancer stem cells can be measured by spheroid assay, measurement of cancer metastasis ability, etc., which will be described later in Examples. That is, the function of cancer stem cells can be said to be a function of forming spheroids, a function of metastasizing cancer, and the like. Moreover, in this specification, the function of a cancer stem cell may be called cancer stem cell property. In some cases, a cell has a function of a cancer stem cell, and a cell has a cancer stem cell property.

As will be described later in Examples, by administering the anticancer agent of the present embodiment, it is possible to suppress the growth of cancer cells, the ability to form spheroids, and the like at the cell level. Moreover, an increase in tumor volume, metastasis of cancer, etc. can be suppressed at the living body level.

In the present specification, cancer stem cells can also be referred to as cancer cells that highly express a cancer stem cell marker. Here, examples of cancer stem cell markers include Oct-4, CD271, JARID1B, and the like. High expression of the cancer stem cell marker means that the expression level of the cancer stem cell marker is higher than that of the control cell. Control cells include normal cells. Alternatively, cancer stem cells can also be said to be cells having the ability to form spheroids or cells having the ability to metastasize and form tumors in vivo.

In the anticancer agent of the present embodiment, the STARD3 / cholesterol change / c-Met pathway inhibitor may be a STARD3 inhibitor, a cholesterol kinetic regulator, or a c-Met inhibitor. May be. Examples of cholesterol kinetic control agents include substances that change the localization of cholesterol, substances that change the abundance of cholesterol, etc., for example, cholesterol transport inhibitors, cholesterol synthesis inhibitors, enzymes involved in steroid metabolism And the like. In the anticancer agent of this embodiment, the cholesterol kinetic regulator is preferably a substance that suppresses the activation of c-Met by changing the kinetics of intracellular cholesterol.

Examples of cholesterol transport inhibitors include U-18666A (CAS number: 3039-71-2). Examples of cholesterol synthesis inhibitors include U-18666A (CAS number: 3039-71-2), fluvastatin (CAS number: 93957-55-2) and the like. Examples of inhibitors of enzymes involved in steroid metabolism include finasteride (CAS number: 98319-26-7).

In the anticancer agent of this embodiment, the STARD3 inhibitor may be a STARD3 expression inhibitor or a substance that suppresses the activity of STARD3. The c-Met inhibitor may be a c-Met expression inhibitor or a substance that decreases the phosphorylation level of c-Met.

Examples of the STARD3 or c-Met expression inhibitor include siRNA, shRNA, miRNA, ribozyme, antisense nucleic acid, and low molecular weight compound.

SiRNA (small interfering RNA) is a low-molecular double-stranded RNA of 21 to 23 base pairs used for gene silencing by RNA interference. The siRNA introduced into the cell binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.

For siRNA, sense strand and antisense strand oligonucleotides are respectively synthesized by a DNA / RNA automatic synthesizer and denatured in an appropriate annealing buffer at 90-95 ° C. for about 1 minute, and then at 30-70 ° C. For about 1-8 hours.

ShRNA (short hairpin RNA) is a hairpin RNA sequence used for gene silencing by RNA interference. shRNA may be introduced into cells by a vector and expressed by U6 promoter or H1 promoter, or an oligonucleotide having shRNA sequence may be synthesized by a DNA / RNA automatic synthesizer and self-annealed in the same manner as siRNA. May also be prepared. The shRNA hairpin structure introduced into the cell is cleaved into siRNA and binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.

MiRNA (microRNA, microRNA) is a functional nucleic acid encoded on the genome and finally converted into a microRNA of about 20 bases through a multi-step production process. miRNAs are classified as functional ncRNAs (non-coding RNAs, non-coding RNAs: generic names for RNAs that are not translated into proteins) and play an important role in life phenomena that regulate the expression of other genes. Yes. By administering miRNA having a specific base sequence to a living body, the expression of STARD3 can be inhibited.

Ribozyme is RNA having catalytic activity. Although some ribozymes have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA. The ribozyme may be a group I intron type, a size of 400 nucleotides or more such as M1 RNA contained in RNaseP, or may be about 40 nucleotides called a hammerhead type, a hairpin type, or the like.

An antisense nucleic acid is a nucleic acid complementary to a target sequence. Antisense nucleic acid inhibits transcription initiation by triplex formation, suppresses transcription by hybridization with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridization with RNA that is being synthesized, Inhibition of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of transition from nucleus to cytoplasm by hybridization with mRNA, capping site and poly (A) addition site Suppression of splicing by hybridization with a protein, suppression of translation initiation by hybridization with a translation initiation factor binding site, suppression of translation by hybridization with a ribosome binding site near the initiation codon, translation region of mRNA and polysome binding site Hybridization outgrowth inhibitory peptide chain by the, by gene silencing due hybridization interaction site between a nucleic acid and a protein, it is possible to suppress the expression of a target gene.

SiRNA, shRNA, miRNA, ribozyme and antisense nucleic acid may contain various chemical modifications in order to improve stability and activity. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue may be substituted with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, and the like. Moreover, you may comprise at least one part with nucleic acid analogs, such as a peptide nucleic acid (PNA).

Further, as described above, the anticancer agent of the present embodiment may be a substance that suppresses the activity of STARD3 or c-Met. Examples of the substance that suppresses the activity of STARD3 or c-Met include a specific binding substance for STARD3, a specific binding substance for c-Met, and the like.

Here, specific binding substances include antibodies, antibody fragments, aptamers, low molecular compounds, and the like. An antibody can be produced, for example, by immunizing an animal such as a mouse with a STARD3 protein or a fragment thereof as an antigen. Alternatively, for example, it can be prepared by screening a phage library. Examples of antibody fragments include Fv, Fab, scFv and the like. The above antibody is preferably a monoclonal antibody. A commercially available antibody may also be used.

An aptamer is a substance having a specific binding ability to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. A nucleic acid aptamer having a specific binding ability to a target substance can be selected by, for example, a systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers having specific binding ability to the target substance can be selected by, for example, the two-hybrid method using yeast.

(67 kDa laminin receptor agonist)
By the way, the inventors have previously observed that 67 kDa laminin receptor (hereinafter sometimes referred to as “67LR”) expression is increased in cancer cells. When a 67LR agonist binds to 67LR, Akt is activated. , Activated endothelial nitric oxide synthase (eNOS), induced nitric oxide (NO) and cGMP production, activated protein kinase Cδ (PKCδ), and produced sphingomyelinase (ASM) Has been shown to be activated and, as a result, induce cell death.

As will be described later in the Examples, when the 67LR agonist is allowed to act on cancer cells, the inventors decrease the expression of STARD3, the expression of c-Met, and the phosphorylation level of c-Met. Was revealed. The inventors have also clarified that when a 67LR agonist is allowed to act on cancer cells, the function of cancer stem cells is inhibited.

Therefore, it can be said that the 67LR agonist is a STARD3 inhibitor. Alternatively, the 67LR agonist can be said to be a STARD3 expression inhibitor. Alternatively, the 67LR agonist can be said to be a c-Met inhibitor. Alternatively, the 67LR agonist can be said to be a c-Met expression inhibitor. Alternatively, a 67LR agonist can be said to be an inhibitor of the c-Met pathway (an inhibitor of c-Met signaling). Alternatively, a 67LR agonist can be said to be an inhibitor of STARD3 / cholesterol change / c-Met pathway.

Examples of 67LR agonists include epigallocatechin-O-gallate (hereinafter sometimes referred to as “EGCG”), which is one of the main catechins contained in green tea, EGCG derivatives such as methylated EGCG, and oolong tea polymerization. Examples include polyphenol, oolong tea polymerization polyphenol derivative, procyanidin, anti-67LR antibody (67LR agonist antibody) and the like.

Oolong tea polymerized polyphenol is a generic name for compounds in which catechins are bound in a complex manner formed by enzymatic reaction or thermal polymerization reaction in a unique method of producing oolong tea called semi-fermentation. For example, catechin dimer, catechin Class of trimers and the like. Examples of catechin dimers include oolong homobisflavans such as oolong homobisflavan A, monodesgaloyl oolong homobisflavan A, oolong homobisflavan B, oolong homobisflavan C, and the like.

[Pharmaceutical composition for cancer treatment]
In one embodiment, the present invention provides a pharmaceutical composition for treating cancer comprising the anticancer agent described above and a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment can also be called a pharmaceutical composition for inhibiting cancer stem cells, a pharmaceutical composition for suppressing cancer metastasis, and the like.

The above-mentioned pharmaceutical composition is administered orally, for example, in the form of tablets, capsules, elixirs, microcapsules, etc., or parenterally in the form of injections, suppositories, external preparations for skin, etc. Can do. More specifically, examples of the external preparation for skin include dosage forms such as ointments and patches.

As the pharmaceutically acceptable carrier, those usually used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, gum tragacanth and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol and glycerin; Examples thereof include adhesives such as rubber adhesives and silicone adhesives.

The pharmaceutical composition may contain an additive. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavoring agents such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid Buffers such as salts and sodium acetate; Solubilizing agents such as benzyl benzoate and benzyl alcohol; Antioxidants; Preservatives and the like.

The pharmaceutical composition can be formulated by appropriately combining the above-mentioned anticancer agents, pharmaceutically acceptable carriers and additives and mixing them in a unit dosage form generally required for pharmaceutical practice.

The dosage of the pharmaceutical composition varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be generally determined, but in the case of oral administration, for example, 0.1-100 mg / kg body weight per dosage unit form What is necessary is just to administer an active ingredient (anticancer agent). In the case of injections, for example, 0.01 to 50 mg of active ingredient may be administered per dosage unit form.

The daily dose of the pharmaceutical composition varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined unconditionally. For example, an effective dose of 0.1-100 mg / kg body weight per day for an adult The components may be administered once a day or divided into 2 to 4 times a day.

[Screening method for anticancer agents]
(First embodiment)
In one embodiment, the present invention includes a step of measuring the expression level of STARD3 in a cell in the presence of a test substance, and the expression level of STARD3 is lower than the expression level in the absence of the test substance. A method for screening for an anticancer agent, comprising the step of determining that the test substance is an anticancer agent. It can also be said that the screening method of this embodiment is a screening method of a cancer stem cell inhibitor or a cancer metastasis inhibitor.

In the screening method of the present embodiment, for example, a cancer cell line can be used as the cell. The cell may be a cancer stem cell or contain a cancer stem cell. Here, the cancer stem cells are the same as those described above, and may be cancer cells that highly express cancer stem cell markers such as Oct-4, CD271, and JARID1B. Or the cell which has spheroid formation ability may be sufficient. The test substance is not particularly limited, and for example, a compound library can be used.

In the screening method of the present embodiment, the expression level of STARD3 may be measured at the gene level by, for example, microarray, real-time PCR, etc., or may be measured at the protein level by ELISA, protein chip, Western blot, or the like.

As described later in Examples, a substance that decreases the expression level of STARD3 can suppress the growth of cancer cells in vivo and in vitro. Moreover, the function of cancer stem cells can be inhibited.

The cancer to be treated by the anticancer agent obtained by the screening method of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

Currently, Wnt / β-catenin pathway, Hedgehog pathway, Notch pathway and the like are known as cancer stem cell maintenance mechanisms. However, any mouse that knocks out these factors is known to be embryonic lethal. Therefore, these factors are considered to have important functions in normal stem cells.

In contrast, STARD3 knockout mice are somewhat more likely to accumulate cholesterol esters when fed with a high fat diet, but there are no differences in survival time, carcinogenesis, and body weight from wild type mice. For this reason, it can be said that STARD3 is a relatively safe drug discovery target.

(Second Embodiment)
In one embodiment, the present invention relates to a step of measuring the activity of STARD3 in the presence of a test substance, and when the activity of STARD3 is reduced compared to the activity in the absence of the test substance, And a step of determining that the test substance is an anticancer agent. It can also be said that the screening method of this embodiment is a screening method of a cancer stem cell inhibitor or a cancer metastasis inhibitor.

According to the screening method of this embodiment, a STARD3 inhibitor can be screened. The screening method of the present embodiment may be performed at the cell level or at a non-cell level (test tube level). When cells are used, the same cells as those in the screening method of the first embodiment described above can be used. The test substance is not particularly limited, and for example, a compound library can be used.

A substance that reduces the activity of STARD3 can suppress the growth of cancer cells in vivo and in vitro. Moreover, the function of cancer stem cells can be inhibited.

The cancer to be treated by the anticancer agent obtained by the screening method of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

(Third embodiment)
In one embodiment, the present invention includes a step of measuring the abundance of cholesterol in a cell in the presence of a test substance, and the abundance of cholesterol is lower than the abundance in the absence of the test substance. A method for screening for an anticancer agent, comprising the step of determining that the test substance is an anticancer agent. It can also be said that the screening method of this embodiment is a screening method of a cancer stem cell inhibitor or a cancer metastasis inhibitor.

As described later in the Examples, cancer cells that suppressed the expression of STARD3 were inhibited from growing, the function of cancer stem cells was inhibited, and the abundance of cholesterol was reduced. Moreover, the function of the cancer stem cell was suppressed in the cancer cell to which the cholesterol kinetic regulator was acted.

In the screening method of the present embodiment, for example, a cancer cell line can be used as the cell. The cell may be a cancer stem cell or contain a cancer stem cell. Here, the cancer stem cells are the same as those described above, and may be cancer cells that highly express cancer stem cell markers such as Oct-4, CD271, and JARID1B. Or the cell which has spheroid formation ability may be sufficient. The test substance is not particularly limited, and for example, a compound library can be used.

The cancer to be treated by the anticancer agent obtained by the screening method of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

(Fourth embodiment)
In one embodiment, the present invention comprises a step of measuring the expression level of c-Met in a cell in the presence of a test substance, and the expression level of c-Met is compared with the expression level in the absence of the test substance. A method of screening for an anticancer agent, comprising the step of determining that the test substance is an anticancer agent when the test substance has decreased. It can also be said that the screening method of this embodiment is a screening method of a cancer stem cell inhibitor or a cancer metastasis inhibitor.

In the screening method of the present embodiment, the same cells as those in the screening method of the first embodiment described above can be used as the cells. Moreover, as a test substance, the thing similar to the thing in the screening method of 1st Embodiment mentioned above can be used.

In the screening method of the present embodiment, the expression level of c-Met may be measured at the gene level by, for example, microarray, real-time PCR, etc., or may be measured at the protein level by ELISA, protein chip, Western blot, or the like.

As described later in Examples, a substance that decreases the expression level of c-Met can suppress the growth of cancer cells. Moreover, the function of cancer stem cells can be inhibited.

The cancer to be treated by the anticancer agent obtained by the screening method of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

(Fifth embodiment)
In one embodiment, the present invention includes a step of measuring the activity of c-Met in the presence of a test substance, and the activity of c-Met is reduced compared to the activity in the absence of the test substance. In some cases, there is provided a method for screening an anticancer agent, comprising the step of determining that the test substance is an anticancer agent. It can also be said that the screening method of this embodiment is a screening method of a cancer stem cell inhibitor or a cancer metastasis inhibitor.

According to the screening method of the present embodiment, c-Met inhibitors can be screened. The screening method of the present embodiment may be performed at the cell level or at a non-cell level (test tube level). When cells are used, the same cells as those in the screening method of the first embodiment described above can be used. The test substance is not particularly limited, and for example, a compound library can be used. As the activity of c-Met, for example, in the case of human or mouse c-Met protein, phosphorylation of the 1234th tyrosine residue (Tyr1234) may be measured.

A substance that decreases the activity of c-Met can suppress the growth of cancer cells in vivo and in vitro. Moreover, the function of cancer stem cells can be inhibited.

The cancer to be treated by the anticancer agent obtained by the screening method of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

[Other Embodiments]
In one embodiment, the invention provides a method of treating cancer comprising administering an effective amount of an inhibitor of STARD3 / cholesterol change / c-Met pathway to a patient in need of treatment. The cancer targeted by the treatment method of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

The treatment method of this embodiment may be referred to as a method of inhibiting cancer stem cells, a method of inhibiting the ability of cancer stem cells to form spheroids, a method of suppressing the expression of cancer stem cell markers of cancer stem cells, a method of suppressing cancer metastasis, and the like. it can.

In the method of the present embodiment, examples of the STARD3 / cholesterol change / c-Met pathway inhibitors include those described above. In addition, one inhibitor of STARD3 / change in cholesterol / c-Met pathway may be used alone, or two or more inhibitors may be used in combination. Moreover, you may use together with the existing anticancer agent.

In one embodiment, the present invention provides an inhibitor of STARD3 / cholesterol change / c-Met pathway for the treatment of cancer. The cancer to be treated by this embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

In this embodiment, “cancer treatment” includes “inhibition of cancer stem cells”, “inhibition of cancer stem cell functions”, “inhibition of spheroid formation ability”, “suppression of expression of cancer stem cell markers”, “cancer metastasis”. In other words, it can be said to be “suppressed”. Further, examples of the inhibitor of STARD3 / change in cholesterol / c-Met pathway include those described above.

In one embodiment, the present invention provides the use of an inhibitor of STARD3 / cholesterol change / c-Met pathway for the manufacture of an anti-cancer agent. The cancer targeted by the anticancer agent of the present embodiment is not particularly limited, and may be melanoma, breast cancer, liver cancer or the like. Further, the above breast cancer may be triple negative breast cancer.

In this embodiment, the “anticancer agent” can also be referred to as “cancer stem cell inhibitor”, “spheroid formation inhibitor”, “cancer stem cell marker expression inhibitor”, “cancer metastasis inhibitor”, and the like. Further, examples of the inhibitor of STARD3 / change in cholesterol / c-Met pathway include those described above.

Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[Materials and methods]
(reagent)
Epigallocatechin gallate (EGCG, model “E4143”), catalase (model “C100”), superoxide dismutase (SOD) (model “S5395”), anti-β-actin antibody (model AS441), cholesterol (model “C8667”) ") Was purchased from Sigma-Aldrich. Alexafluoro 488 goat anti-rabbit IgG antibody (type “A-11034”) was purchased from Invitrogen. Anti-STARD3 antibody was purchased from Proteintech. Anti-Oct-4 antibody, anti-CD271 antibody, anti-JARID1B antibody and anti-Met antibody were purchased from Cell Signaling Technology. U-18666A (model “100009869”) was purchased from Cayman Chemical Company. Fluvastatin was purchased from LKT Laboratories. Finasteride was purchased from Tokyo Chemical Industry Co., Ltd. Recombinant mouse HGF (model “550-84491”) was purchased from R & D Systems.

(Cell culture)
Mouse melanoma cell line B16, human melanoma cell line MeWo, A375, human breast cancer cell line MDA-MB-231, mouse breast cancer cell line 4T1, liver cancer cell line HepG2 in DMEM medium supplemented with 5% or 10% fetal calf serum (FCS) The culture was maintained at 37 ° C. under 5% CO ₂ with water vapor saturation and maintained in the logarithmic growth phase.

(Genetic suppressor elements (GSE) library)
In GSE library (MFL-ESP), mouse embryo cDNA (model “ML8000BB”, Clontech) was fragmented with restriction enzymes EcoRI and SphI and introduced into a pLPCX-modified retrovirus vector. pVSV-G vector was purchased from Takara Bio Inc. pTargetT ™ Mammalian Expression Vector System was purchased from Promega.

(GSE library introduction and selection by EGCG)
EcoPack-293 and AmphoPack-293 packaging cells were each adjusted to 2.0 × 10 ⁵ cells / mL, seeded in 5 mL dishes, and cultured in DMEM medium containing 10% FBS. The next day, 1.0 μg / μL MFL-ESP 3 μL, 1.0 μg / μL 3 μL of pVSV-G, and 6 μL of FuGENE6 transfection reagent (Roche) were introduced into each packaging cell. The next day, the culture supernatant of the packaging cells was passed through a filter (0.22 μm), and polybrene (hexadimethyline bromide, Sigma-Aldrich) was added to a final concentration of 8 μg / mL, and then 1 × 10 ⁴ cells the day before. Adjusted to / mL, seeded in a 5 mL dish, added to B16 cells cultured in DMEM medium containing 5% FBS, and infected with the virus in the culture supernatant. To the packaging cells, fresh DMEM medium containing 10% FBS was added, and the culture was continued. Medium change was performed 4 times every 12 hours. B16 cells after infection were adjusted to 1 × 10 ⁴ cells / mL, seeded at 0.1 mL / well in a 96-well plate, and recovered for 24 hours in DMEM medium containing 2% FBS. After recovery culture, selection was performed for about 1 month in 1% FBS-containing DMEM medium supplemented with EGCG having a final concentration of 50 μM.

(Quantitative RT-PCR (qRT-PCR))
RNA was extracted from the cells using TRIzol reagent (Invitrogen), and cDNA was synthesized using PrimeScript RT reagent kit (Takara Bio Inc.). Quantitative PCR was performed using the obtained cDNA. For the quantitative PCR, Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc.) was used. The base sequence of the sense primer for amplification of mouse Micropalmia Transduction Factor (MITF) is shown in SEQ ID NO: 3, and the base sequence of the antisense primer is shown in SEQ ID NO: 4. The base sequence of the sense primer for amplifying mouse tyrosinase is shown in SEQ ID NO: 5, and the base sequence of the antisense primer is shown in SEQ ID NO: 6. Further, the base sequence of the sense primer for amplifying β-actin is shown in SEQ ID NO: 7, and the base sequence of the antisense primer is shown in SEQ ID NO: 8.

(Western blot)
Lysis buffer (Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-X100, 1 mM EDTA, 50 mM NaF, 30 mM Na ₄ P ₂ O ₇ , 1 mM phenylmethylsulfonyl fluoride (PMSF), 2.0 μM / mL) After suspending the cells in aprotinin and centrifuging at 500 × g for 5 minutes, the supernatant was mixed with SDS-PAGE sample buffer (0.057 M Tris-HCl, pH 6.8, 9.1% glycerol, 1.8% SDS, 0.02% bromophenol blue, 0.65M 2-mercaptoethanol) and diluted for 2 minutes to prepare a sample for SDS-PAGE.

Subsequently, the sample was subjected to electrophoresis using an SDS-PAGE gel. Subsequently, the protein in the gel was transferred to a nitrocellulose membrane (Schleicher & Schuell) at a voltage of 100 V for 60 minutes. After blocking with 2.5% BSA-TTBS (Tris buffered saline containing 0.1% Tween20; 20 mM Tris-HCl, pH 7.6) for 1 hour at room temperature, the primary antibody was diluted with 2.5% BSA-TTBS. The reaction was allowed to proceed overnight at 4 ° C. After washing with TTBS, a secondary antibody solution diluted with 2.5% BSA-TTBS was reacted for 1 hour. Subsequently, after washing 3 times with TTBS, a color reaction was performed using Enhanced Chemiluminescence Reagent (ECL) Advance Western Bleaching Detection (GE Healthcare), and the luminescence intensity was analyzed.

(Immunohistochemical staining using tissue from human melanoma and breast cancer patients)
A human melanoma tissue array (model “ME802”, US BIOMAX) and a breast cancer tissue array (model “BR1503”, US BIOMAX) were immersed in xylene for 10 minutes, and then immersed in new xylene and allowed to stand for 10 minutes. Subsequently, 100%, 95%, and 70% ethanol were prepared, the slide glass was immersed for 5 minutes in descending order of concentration, and finally deparaffinized by being immersed in a phosphate buffer solution (PBS) for 5 minutes. .

Subsequently, 10 mM sodium citrate (pH 6.0) was placed in a beaker, the slide glass was immersed, and the beaker was placed in a pressure cooker containing deionized water. After pressurizing, heat treatment was performed for 10 minutes, and the slide glass was taken out and then air-cooled for 30 minutes. Subsequently, after blocking with 5% BSA for 1 hour at room temperature, the target antibody was replaced with diluted 5% BSA and reacted at 4 ° C. overnight. Subsequently, after washing with deionized water, 5% BSA diluted with Alexafluoro488 anti-rabbit IgG antibody was reacted at room temperature for 1 hour to label the target protein. Further, after washing with deionized water, it was replaced with 5% BSA containing DAPI for staining nuclei and allowed to stand at room temperature for about 5 minutes. Finally, after washing with deionized water, a cover glass was placed and observed with a fluorescence microscope.

(Suppression of gene expression by RNA interference method)
Non-targeting Scr-shRNA (model “SHC016”, hereinafter sometimes referred to as “Scr-shRNA”) and shRNAs targeting STAR3 (human: model “TRCN000000155584”, mouse: model “TRCN0000105330”, hereinafter “STRD3” -ShRNA ".) Lentiviral vector was purchased from Sigma-Aldrich and used. The target sequence of the type “TRCN0000155584” is shown in SEQ ID NO: 1 and the target sequence of the type “TRCN0000105330” is shown in SEQ ID NO: 2. Vector purification was performed according to the manual.

As transient knockdown siRNA for STARD3, Ambion In Vivo Pre-designed Negative control siRNA # 1 (model “# 4457289”, hereinafter may be referred to as “Scr-siRNA”) and Ambion InreVivDreInVidInPrevSTP siRNA (model “# s81858”, hereinafter sometimes referred to as “STARD3-siRNA”) was purchased from Thermo Fisher Scientific and used.

As transient knockdown siRNA against c-Met, Mission negative control siRNA (model “SIC-001”, hereinafter sometimes referred to as “Ctl-siRNA”) and siRNA targeting c-Met (model “SASI_Mm01_00095875”) Hereinafter, it may be referred to as “c-Met-siRNA”) was purchased from Sigma-Aldrich and used.

(Receptor tyrosine kinase (RTKs) activity measurement)
Cells were pre-cultured in serum-free medium for 24 hours, then replaced with 10% FBS-DMEM medium, and incubated at 37 ° C. for 30 minutes to induce phosphorylation of RTKs. Thereafter, phosphorylation levels of various RTKs were measured using a phosphor-RTK array kit (R & D Systems).

(Spheroid assay)
Cells are diluted to 2000 cells / well, seeded in Corning Ultra-Low Attachment Surface 24 well plates (model “3473”, Corning), EGF (20 ng / mL, BD Biosciences), bFGF (10 ng). / ML, BD Biosciences) and B27 (1:50 dilution, Invitrogen) were cultured in serum-free DMEM medium for 21 days. Thereafter, the number of spheroids was counted.

(Animal experimentation)
<Measurement of tumor volume and survival rate>
Five-week-old female C57BL / 6J mice were scrambled with scrambled shRNA (Scr-shRNA) or STAT3-repressed shRNA (STARD3-shRNA) stably introduced B16 cells at the back of 5 × 10 ⁵ cells / mouse. Transplanted subcutaneously. Tumor volume was measured once every two days. The tumor volume was calculated by the following formula (1).
Tumor volume = length × width ^2/2 (1)
Survival curves were prepared using the Kaplan-Meier method and compared by the two-way ANOVA test.

<< Examination of lung metastasis 1 >>
Five-week-old female C57BL / 6J mice were scrambled shRNA (Scr-shRNA) or STAT3-repressed shRNA (STARD3-shRNA) stably introduced B16 cells at 5 × 10 ⁵ cells / mouse. Transplanted into a vein. Three weeks later, the lungs were removed and observed for lung metastasis.

<< Examination of lung metastasis 2 >>
4-week-old female BALB / c mice were transplanted subcutaneously on the back with 4T1 cells at 1 × 10 ⁵ cells / 200 μL PBS. Every week, 15 nM / mouse scrambled siRNA (Scr-siRNA) or siRNA that suppresses the expression of STARD3 (STARD3-siRNA) was administered together with the same amount of atelocollagen in and around the subcutaneous tumor. Tumor volume and body weight were measured once every two days. On the last day, assessment of metastasis in the lung and ALT / AST activity in serum were measured.

(Measurement of cholesterol level in whole cells)
1 × 10 ⁶ B16 cells or MeWo cells stably introduced with Scramble-shRNA or STARD3-shRNA / sample were mixed with 200 μL of an organic mixture (chloroform: isopropanol: NP-40 = 7: 11: 1.0) using a microhomogenizer. Extracted in 1). Subsequently, the mixture was centrifuged at 15,000 × g for 10 minutes, the supernatant was transferred to a new tube, and the lipid containing cholesterol was dried with a speed-back centrifugal concentration system (Thermo Fisher Scientific). Subsequently, the dried lipid was dissolved in 100 μL Cholesterol Assay Buffer, mixed uniformly with a vortex mixer, and 25 μL of the extract was used for total cholesterol and cholesterol Este Colorimetry / Fluorometric Assay Kit (Amount of Biochemical Assay Kit) Was measured.

[Experimental Example 1]
(Identification of STARD3 by Genetic suppressor elements (GSE) method)
The inventors previously revealed that epigallocatechin gallate (EGCG), which is a green tea catechin, has a cancer cell growth inhibitory action. Therefore, an attempt was made to identify a factor essential for the cell growth inhibitory action by EGCG. Specifically, the GSE library was introduced into B16 cells, which are a mouse melanoma cell line, and selected for about 1 month in an EGCG-containing medium. Thereby, the cell which shows tolerance with respect to the cell growth inhibitory effect of EGCG was acquired. Since melanoma stem cells are in a dedifferentiated state and are known to have a reduced ability to synthesize melanin, cells in a differentiated state were selected from resistant cells using the amount of pigment as an index. When the gene fragment introduced into this cell was analyzed by sequencing, StAR-related lipid transfer domain containing 3 (STARD3) was identified. FIG. 1 is a diagram illustrating a process of identifying a STARD3 gene.

[Experiment 2]
(Inhibition of STARD3 protein expression by EGCG)
The effect of EGCG on the expression of STARD3 protein was examined by Western blot. Specifically, B16 cells were cultured for 0, 24, 48, 72, and 96 hours in a medium containing EGCG (5 units / mL SOD, 200 units / mL catalase, 1% FCS) with a final concentration of 5 μM. The expression of STARD3 protein was detected by Western blot. As a control, β-actin protein was detected.

FIG. 2 (a) is a photograph showing the results of Western blotting. Moreover, FIG.2 (b) is the graph which digitized the result of Fig.2 (a).

As a result, it has been clarified that the expression level of the STARD3 protein is decreased depending on the treatment time by the treatment with EGCG.

[Experiment 3]
(Promotion of melanoma differentiation by suppression of expression specific to STARD3)
In order to examine the involvement of STARD3 in melanoma stem cells, B16 cells in which the expression of STARD3 was suppressed by RNA interference were prepared, and dye synthesis was evaluated.

FIGS. 3 (a) and 3 (b) are photomicrographs of control B16 cells (Scr-shRNA) and B16 cells (STARD3-shRNA) in which the expression of STARD3 was suppressed, respectively. As a result, it was revealed that the B16 cells that suppressed the expression of STARD3 were darker than the control.

Subsequently, RNA was collected from each cell, and the expression levels of Microphthalmia Transduction Factor (MITF) and tyrosinase gene, which are melanoma differentiation markers, were measured by quantitative PCR. In addition, the expression level of β-actin gene was measured as an internal standard.

FIG. 3 (c) is a graph showing the results of quantitative PCR. As a result, it was revealed that the expression levels of MITF and tyrosinase were significantly higher in the B16 cells in which the expression of STARD3 was suppressed compared to the control. This result shows that suppression of STARD3 expression promotes differentiation of melanoma cells.

[Experimental Example 4]
(Investigation of the effect of suppression of STARD3 expression on the expression of cancer stem cell markers)
Oct-4 has been shown to be a cancer stem cell marker in various cancers including melanoma. CD271 and JARID1B have been reported as melanoma stem cell markers. Thus, shRNA (STARD3-shRNA) that suppresses the expression of STARD3 was introduced into mouse melanoma cell line B16 and human melanoma cell line MeWo, respectively, and the expression levels of these markers were examined by Western blot.

4 (a) and 4 (b) are photographs showing the results of Western blotting. FIG. 4 (a) shows the results for B16 cells, and FIG. 4 (b) shows the results for MeWo cells. As a result, it was revealed that the expression levels of Oct-4, CD271, and JARID1B were decreased by the suppression of STARD3 expression in any cell. This result indicates that suppression of STARD3 expression inhibits melanoma stem cell function.

[Experimental Example 5]
(Investigation of the effect of suppression of STARD3 expression on spheroid formation ability)
Spheroid-forming ability is one index of cancer stem cell function. Therefore, shRNA (STARD3-shRNA) that suppresses the expression of STARD3 was introduced into B16 cells and MeWo cells, respectively, and the spheroid-forming ability of these cells was measured. As a control, cells into which scrambled shRNA (Scr-shRNA) was introduced were used.

FIGS. 5A to 5D are photographs and graphs showing the results of the spheroid assay. FIG. 5 (a) is a representative photomicrograph of B16 cells. FIG.5 (b) is a graph which shows the result of having measured the number of spheroids of B16 cell. FIG. 5C is a representative photomicrograph of MeWo cells. FIG.5 (d) is a graph which shows the result of having measured the number of spheroids of the MeWo cell.

As a result, it was revealed that spheroid formation ability was suppressed in any cell. This result further supports that suppression of STARD3 expression inhibits melanoma stem cell function.

[Experimental Example 6]
(Examination of STARD3 expression in tumor tissue of melanoma patients 1)
The expression of STARD3 in normal skin tissue and tumor tissue of melanoma patients was evaluated by immunohistochemical staining. FIG. 6A is a representative fluorescence micrograph showing the results of immunohistochemical staining. FIG. 6B is a graph summarizing the results of immunohistochemical staining. As a result, it was revealed that the expression level of STARD3 was higher in the tumor tissue of the melanoma patient than in the normal skin tissue.

[Experimental Example 7]
(Examination of STARD3 expression in tumor tissue of melanoma patients 2)
Based on the accession number GSE3189 in the microarray database GEO, the expression level of STARD3 mRNA was compared between normal human skin tissue and tumor tissue of melanoma patients. FIG. 7 is a graph showing the examination results. As a result, it was revealed that STARD3 expression level is high in tumor tissues of melanoma patients.

[Experimental Example 8]
(Inhibition of melanoma cell proliferation by suppression of STARD3 expression)
ShRNA (STARD3-shRNA) that suppresses the expression of STARD3 was introduced into the mouse melanoma cell line B16 cells, the human melanoma cell line A375 cells, and MeWo cells, respectively, and cell proliferation was examined. Each cell into which scrambled shRNA (Scr-shRNA) was introduced was used as a control.

FIGS. 8A to 8C are graphs showing the measured cell growth curves. FIG. 8 (a) shows the results for B16 cells, FIG. 8 (b) shows the results for A375 cells, and FIG. 8 (c) shows the results for MeWo cells. As a result, it was revealed that cell proliferation was remarkably suppressed by suppressing the expression of STARD3 in any cell.

[Experimental Example 9]
(Inhibition of melanoma tumor growth by suppression of STARD3 expression)
B16 cells into which shRNA that suppresses the expression of STARD3 (STARD3-shRNA) was introduced were implanted subcutaneously in the back of 5-week-old female C57BL / 6J mice, and the survival period was evaluated. As a control, mice transplanted with B16 cells introduced with scrambled shRNA (Scr-shRNA) were used. In addition, the tumor volume of each mouse was measured once every two days. Mice were euthanized when the tumor volume reached 4000 mm ³ .

FIG. 9 (a) is a representative photograph of a mouse (STARD3-shRNA) and a control mouse (Scr-shRNA) transplanted with B16 cells in which the expression of STARD3 is suppressed. FIG. 9B is a representative photograph showing a tumor tissue excised from each mouse. Moreover, FIG.9 (c) is a graph which shows the measurement result of the tumor volume of each mouse | mouth. FIG. 9D is a graph showing the survival curve of each mouse.

As a result, it was revealed that tumor growth was significantly suppressed in mice transplanted with B16 cells in which the expression of STARD3 was suppressed as compared with control mice. In addition, it was revealed that the survival period of the mice transplanted with B16 cells in which the expression of STARD3 was suppressed was significantly prolonged as compared with the control mice.

[Experimental Example 10]
(Inhibition of lung metastasis of melanoma cells by suppression of STARD3 expression)
B16 cells into which shRNA that suppresses the expression of STARD3 (STARD3-shRNA) was introduced were transplanted from the tail vein of mice to induce lung metastasis. As a control, mice transplanted with B16 cells introduced with scrambled shRNA (Scr-shRNA) were used. Subsequently, lungs were removed from each mouse after 3 weeks, and the effect of STARD3 on pulmonary metastasis of melanoma was examined. FIG. 10 is a photograph of the lungs removed from each mouse.

As a result, it became clear that lung metastasis was not observed in mice transplanted with B16 cells in which the expression of STARD3 was suppressed. From this result, it became clear that suppression of STARD3 expression inhibits lung metastasis of melanoma cells.

[Experimental Example 11]
(Effects of STARD3 expression on the prognosis of melanoma patients)
Based on the accession number GSE65904 in the microarray database GEO, the relationship between the expression level of STARD3 in melanoma patients and the survival time was analyzed. Based on the expression level of STARD3, the patients were divided into two groups, and the survival time was analyzed based on the Kaplan-Meier method.

FIG. 11 is a graph showing the examination results. As a result, the group with high expression level of STARD3 was calculated to have a survival period of 751 days, and the group with low expression level of STARD3 was calculated to have a survival period of 1164 days. From this result, it became clear that patients with high expression levels of STARD3 have a poor prognosis.

[Experimental example 12]
(Reduction of cholesterol level by suppression of STARD3 expression)
The relationship between the expression of STARD3 and the amount of cholesterol was examined. Specifically, the amount of cholesterol in B16 cells and MeWo cells into which shRNA that suppresses the expression of STARD3 (STARD3-shRNA) was introduced was measured. As controls, B16 cells and MeWo cells into which scrambled shRNA (Scr-shRNA) was introduced were used.

12 (a) and 12 (b) are graphs showing measurement results. FIG. 12 (a) shows the results for B16 cells, and FIG. 12 (b) shows the results for MeWo cells. As a result, it was clarified that the cholesterol level of the whole cell was significantly reduced by suppressing the expression of STARD3 in any cell.

[Experimental Example 13]
(Examination of the effect of cholesterol kinetic regulators on spheroid formation ability)
A melanoma cell line was cultured in the presence of a cholesterol kinetic regulator, and spheroid-forming ability was evaluated. U-18666A, finasteride, and fluvastatin were used as cholesterol kinetic regulators.

FIGS. 13A to 13C are graphs showing the results of the spheroid assay. FIG. 13 (a) shows the result of spheroid assay performed by adding 0, 1.25 and 2.5 μM U-18666A to the medium of B16 cells. FIG. 13 (b) shows the result of spheroid assay performed by adding 0, 1.25 and 2.5 μM U-18666A to the MeWo cell medium. FIG. 13 (c) shows the result of spheroid assay performed by adding 2.5 μM finasteride or 2.5 μM fluvastatin to the medium of B16 cells. As a control, B16 cells to which no cholesterol kinetic regulator was added were used.

As a result, it has been clarified that the addition of each cholesterol kinetic regulator inhibits the spheroid formation ability of B16 cells and MeWo cells. This result indicates that cholesterol kinetic regulators inhibit melanoma stem cell function.

[Experimental Example 14]
(Examination of the effects of cholesterol kinetic regulators on the expression of cancer stem cell markers)
B16 cells were cultured in the presence of a cholesterol kinetic regulator, and the expression level of Oct-4, a cancer stem cell marker, was examined by Western blot. The expression level of β-actin was examined as an internal standard. U-18666A, finasteride, and fluvastatin were used as cholesterol kinetic regulators.

FIGS. 14 (a) to 14 (d) are photographs showing the results of Western blotting. FIG. 14A shows the result of U-18666A. FIG. 14B is a graph in which the result of FIG. FIG. 14 (c) is a photograph showing the results of Western blotting of B16 cells cultured in a medium supplemented with 2.5 μM finasteride or 2.5 μM fluvastatin. As a control, B16 cells to which no cholesterol kinetic regulator was added were used. FIG. 14D is a graph in which the result of FIG. 14C is digitized.

As a result, it was revealed that the expression level of Oct-4 decreased in the presence of any cholesterol kinetic regulator. This result further supports that cholesterol kinetic regulators inhibit melanoma stem cell function.

[Experimental Example 15]
(Enhancement of melanoma stem cell function by cholesterol)
B16 cells were cultured in a medium supplemented with cholesterol, and a spheroid assay was performed. FIG. 15 is a graph showing the results of the spheroid assay. As a result, it was revealed that the ability of B16 cells to form spheroids is enhanced in the presence of cholesterol. This result indicates that cholesterol enhances melanoma stem cell function.

[Experimental Example 16]
(Disappearance of melanoma stem cell function enhancing action of cholesterol by suppressing expression of STARD3)
Cholesterol was added to the culture medium of B16 cells introduced with shRNA that suppresses the expression of STARD3 (STARD3-shRNA) and B16 cells introduced with control scrambled shRNA (Scr-shRNA), and spheroid assay was performed. FIG. 16 is a graph showing the results of the spheroid assay.

As a result, in the control B16 cells, the ability to form spheroids was enhanced by the addition of cholesterol. On the other hand, in B16 cells in which the expression of STARD3 was suppressed, it was revealed that no change was observed in spheroid formation ability even when cholesterol was added. From this result, it became clear that suppression of STARD3 expression disappears enhancement of melanoma stem cell function by cholesterol.

[Experimental Example 17]
(Inhibition of receptor tyrosine kinase (RTKs) activity by suppression of STARD3 expression)
It was investigated whether suppression of STARD3 expression affects the activity of RTKs. The phosphorylation level of RTKs in B16 cells stably transfected with shRNA (STARD3-shRNA) or scrambled shRNA (Scr-shRNA) that suppresses the expression of STARD3 was evaluated using a phosphor-RTK array kit (R & D Systems).

FIG. 17 is a graph showing experimental results. As a result, it has been clarified that there are RTKs whose activities are reduced by suppressing the expression of STARD3. In particular, the decrease in c-Met (HGFR) activity was significant.

[Experiment 18]
(Inhibition of c-Met by suppressing the expression of STARD3)
Phosphorylation of the 1234th tyrosine residue (Tyr1234) of c-Met protein and c-Met protein in B16 cells stably transfected with shRNA (STARD3-shRNA) or scrambled shRNA (Scr-shRNA) that suppresses the expression of STARD3 The expression of was examined by Western blot.

FIG. 18 (a) is a photograph showing the results of Western blotting. FIG. 18B is a graph in which the result of FIG. As a result, it was revealed that the suppression of STARD3 expression decreased phosphorylation of c-Met Tyr1234 and the expression level of c-Met.

[Experimental Example 19]
(Inhibition of c-Met by cholesterol transport inhibitor U-18666A)
B16 cells were cultured for 96 hours in the presence of 0, 1, and 2.5 μM U-18666A, and phosphorylation of c-Met Tyr1234 and expression of c-Met were examined by Western blot.

FIG. 19 (a) is a photograph showing the results of Western blotting. FIG. 19 (b) is a graph obtained by quantifying the result of the expression level of c-Met in FIG. 19 (a). As a result, it was revealed that the phosphorylation level of c-Met Tyr1234 and the expression level of c-Met decreased in the presence of U-18666A. From this result, it was revealed that cholesterol controls the activity of c-Met.

[Experiment 20]
(Inhibition of melanoma stem cell marker expression by suppressing c-Met expression)
The effect of c-Met on melanoma stem cells was examined. Specifically, 96 hours after introduction of siRNA that suppresses c-Met expression (c-Met-siRNA) or scrambled siRNA (Ctl-siRNA) into B16 cells, the expression of Oct-4 was examined by Western blot. did.

FIG. 20 (a) is a photograph showing the results of Western blotting. FIG. 20B is a graph in which the result of FIG. As a result, it was revealed that the expression level of Oct-4 was decreased by suppressing the expression of c-Met. This result indicates that c-Met positively regulates Oct-4 expression.

[Experiment 21]
(Enhancement of melanoma stem cell function by hepatocyte growth factor HGF)
The effect of HGF, which is a ligand of c-Met, on melanoma stem cell function was examined. Specifically, B16 cells were stimulated for 30 minutes in the presence of 0, 10, 25, and 50 ng / mL HGF, and c-Met Tyr1234 phosphorylation and Oct-4 expression levels were examined by Western blot. In addition, B16 cells were cultured in the presence of 50 ng / mL HGF, and spheroid assay was performed.

FIG. 21 (a) is a photograph showing the results of Western blotting. FIG. 21B is a graph in which the result of FIG. FIG. 21 (c) is a graph showing the results of the spheroid assay. As a result, it has been clarified that phosphorylation of Tyr1234 of c-Met is promoted and the expression level of Oct-4 increases in the presence of HGF. In addition, it has been clarified that the spheroid-forming ability of B16 cells is enhanced in the presence of HGF. The above results revealed that c-Met activation enhances melanoma stem cell function.

[Experimental example 22]
(STARD3 is highly expressed in triple negative breast cancer tumor tissue compared to normal human breast tissue)
The expression level of STARD3 in normal breast tissue and triple negative breast cancer tumor tissue was compared by immunohistochemical staining. Triple negative breast cancer is a breast cancer in which estrogen receptor (ER), progesterone receptor (PgR), and HER2 are all negative, and is known to have a poor prognosis.

FIG. 22 is a fluorescence micrograph showing the results of immunohistochemical staining. As a result, it was revealed that STARD3 expression level is high in triple negative breast cancer tumor tissues.

[Experimental example 23]
(Effects of cholesterol kinetic regulators on the expression of c-Met in triple negative breast cancer cell lines)
MDA-MB-231 cells, a human triple negative breast cancer cell line, were cultured in the presence of a cholesterol kinetic regulator, and c-Met expression was examined by Western blot. The expression level of β-actin was examined as an internal standard. 0 and 5 μM U-18666A were used as cholesterol kinetic regulators.

FIG. 23 (a) is a photograph showing the results of Western blotting. FIG. 23B is a graph in which the result of FIG. As a result, it was revealed that the expression level of c-Met in the triple negative breast cancer cell line decreased in the presence of U-18666A. From this result, it was revealed that cholesterol also controls the activity of c-Met in triple negative breast cancer cells.

[Experimental example 24]
(Effects of cholesterol transport inhibitors on spheroid formation ability of triple-negative breast cancer cell lines)
MDA-MB-231 cells, a human triple negative breast cancer cell line, were cultured in the presence of a cholesterol kinetic regulator and spheroid assay was performed. As cholesterol kinetic regulators, 0, 1.25 and 2.5 μM U-18666A were used.

FIG. 24 is a graph showing the results of the spheroid assay. As a result, in the presence of U-18666A, it was revealed that the ability of MDA-MB-231 cells to form spheroids is inhibited. This result shows that cholesterol kinetic regulators inhibit cancer stem cell function of triple negative breast cancer.

[Experiment 25]
(Inhibition of lung metastasis of triple negative breast cancer cells by local administration of siRNA targeting STARD3)
A mouse triple negative breast cancer cell line, 4T1, was implanted subcutaneously in the back of the mouse to form tumors. Subsequently, 15 nM / mouse scrambled siRNA (Scr-siRNA) or siRNA that suppresses the expression of STARD3 (STARD3-siRNA) was administered together with the same amount of atelocollagen in and around the subcutaneous tumor every week. Four weeks later, the lungs were removed, and the number of lung metastasis nodules and lung weight were measured as indicators of lung metastasis. Moreover, in order to evaluate the toxicity by administration of siRNA, the ALT / AST activity in serum was measured.

FIG. 25 (a) is a representative photograph of the lung removed from the mouse. Arrowheads indicate lung metastasis nodules. FIG. 25 (b) is a graph showing the measurement results of the number of lung metastatic nodules. FIG. 25 (c) is a graph showing the measurement results of lung weight. FIG. 25 (d) is a graph showing the measurement results of AST activity. FIG. 25 (e) is a graph showing the measurement results of ALT activity. In FIGS. 25D and 25E, “ns” indicates that there is no significant difference.

As a result, it was clarified that the mice administered with STARD3-siRNA had a small number of lung metastasis nodules and an increase in lung weight was also suppressed. In addition, no change in AST / ALT activity due to administration of STARD3-siRNA was observed. From the above results, it became clear that administration of STARD3-siRNA can suppress the metastasis of triple negative breast cancer cells without seriously affecting the living body.

[Experiment 26]
(Examination of STARD3 expression in liver cancer tissue)
Based on the accession number GSE54236 in the microarray database GEO, the expression level of STARD3 mRNA was compared between normal liver tissue and tumor tissue of a liver cancer patient. FIG. 26 is a graph showing the examination results. As a result, it was revealed that the expression level of STARD3 was higher in the tumor tissue of the liver cancer patient than in the normal liver tissue.

[Experiment 27]
(Examination of the effect of cholesterol kinetic regulators on liver cancer stem cell function 1)
The liver cancer cell line HepG2 was cultured in the presence of a cholesterol kinetic regulator, and the expression levels of c-Met and the cancer stem cell marker Oct-4 were examined by Western blot. The expression level of β-actin was examined as an internal standard. As cholesterol kinetic regulators, 0, 1.25 and 2.5 μM U-18666A were used.

FIG. 27 is a photograph showing the results of Western blotting. As a result, it was revealed that the expression levels of c-Met and Oct-4 in the liver cancer cell line decreased in the presence of the cholesterol kinetic regulator. From this result, it was revealed that cholesterol kinetic regulators inhibit liver cancer stem cell function.

[Experiment 28]
(Examination of the effects of cholesterol kinetic regulators on liver cancer stem cell function 2)
Liver cancer cell line HepG2 was cultured in the presence of a cholesterol kinetic regulator and spheroid assay was performed. As cholesterol kinetic regulators, 0, 1.25 and 2.5 μM U-18666A were used.

FIG. 28 is a graph showing the results of the spheroid assay. As a result, it has been clarified that the ability of HepG2 cells to form spheroids is inhibited in the presence of a cholesterol kinetic regulator. This result indicates that cholesterol kinetic regulators inhibit liver cancer stem cell function.

[Experimental example 29]
(Reduction of STARD3 expression by 67LR agonist)
A 67LR agonist was added to the medium of B16, a mouse melanoma cell line, MDA-MB-231, a human breast cancer cell line, and 4T1, a mouse breast cancer cell line, and the effect on STARD3 expression was examined. EGCG was used as the 67LR agonist.

Mouse melanoma cell line B16 was seeded at a cell density of 1 × 10 ⁴ cells / mL in a 24-well plate at 1 mL / well and pre-cultured in 5% FBS-DMEM medium for 24 hours. Thereafter, the cells were cultured for 96 hours in a medium (5 units / mL SOD, 200 units / mL catalase, 1% FBS) containing EGCG having a final concentration of 0, 1, 10 μM.

Subsequently, each cell was dissolved in a cell lysis buffer and collected, and the expression level of STARD3 protein was measured by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 30 (a) is a photograph showing the results of Western blotting and a graph in which the results are digitized. In the graph of FIG. 30 (a), the results are expressed as mean ± standard deviation, “**” indicates that there is a significant difference at P <0.01 in Dunnett's test, and “ns” Represents no significant difference.

In addition, human breast cancer cell line MDA-MB-231 was seeded at a cell density of 1 × 10 ⁴ cells / mL in a 24-well plate at 1 mL / well and pre-cultured in 10% FCS-RPMI medium for 24 hours. Thereafter, the cells were cultured for 96 hours in a medium (5 units / mL SOD, 200 units / mL catalase, 1% FCS) containing EGCG having a final concentration of 0, 1, 5, 10 μM.

Subsequently, each cell was dissolved in a cell lysis buffer and collected, and the expression level of STARD3 protein was measured by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 30B is a photograph showing the results of Western blotting and a graph in which the results are digitized. In the graph of FIG. 30 (b), the result is expressed as an average value ± standard deviation, and “***” indicates that there is a significant difference at P <0.001 in Dunnett's test.

In addition, mouse breast cancer cell line 4T1 was seeded at a cell density of 1 × 10 ⁴ cells / mL in a 24-well plate at 1 mL / well and pre-cultured in 10% FCS-RPMI medium for 24 hours. Thereafter, the cells were cultured in a medium containing EGCG having a final concentration of 0, 1, 5, 10 μM (5 units / mL SOD, 200 units / mL catalase, 1% FCS) for 72 hours.

Subsequently, each cell was dissolved in a cell lysis buffer and collected, and the expression level of STARD3 protein was measured by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 30 (c) is a photograph showing the results of Western blotting and a graph in which the results are digitized. In the graph of FIG. 30 (c), the result is expressed as an average value ± standard deviation, “**” indicates that a significant difference exists at P <0.01 in Dunnett's test, and “***” indicates P <0.001 indicates that there is a significant difference.

As a result, it was revealed that the expression level of STARD3 in the mouse melanoma cell line B16, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 decreases when the 67LR agonist is allowed to act. This result indicates that the 67LR agonist is a STARD3 expression inhibitor (expression suppressor).

[Experiment 30]
(Examination of the effect of 67LR agonist on spheroid formation ability)
Spheroid-forming ability is one index of cancer stem cell function. Therefore, a 67LR agonist was added to the culture medium of mouse melanoma cell line B16, mouse breast cancer cell line 4T1, and human liver cancer cell line HepG2, and the influence on the spheroid formation ability of these cells was examined. EGCG was used as the 67LR agonist.

Mouse melanoma cell line B16 was seeded at 100 μL / well in a 96-well low adhesion plate (Corning) at a cell density of 2000 cells / mL and added with EGCG at a final concentration of 0 or 10 μM, 20 ng / mL EGF, 20 ng / mL After culturing in serum-free DMEM medium containing bFGF, B27 (1:50 dilution, Invitrogen), 5 units / mL SOD, 200 units / mL catalase, the number of spheroids was measured (n = 6).

FIG. 31 (a) is a graph showing the results of the spheroid assay. In FIG. 31 (a), the result is expressed as an average value ± standard deviation, and “**” indicates that there is a significant difference at P <0.01 in Dunnett's test.

In addition, the mouse breast cancer cell line 4T1 was seeded at a cell density of 2000 cells / mL in a 24-well low adhesion plate (Corning) at 1 mL / well, and EGCG was added at a final concentration of 0, 1, 5, 10 μM, 20 ng / After culturing in serum-free DMEM medium containing mL EGF, 20 ng / mL bFGF, B27 (1:50 dilution, Invitrogen), 5 units / mL SOD, 200 units / mL catalase, the number of spheroids was measured ( n = 3).

FIG. 31 (b) is a graph showing the results of the spheroid assay. In FIG. 31 (b), the result is expressed as an average value ± standard deviation, “*” indicates that there is a significant difference at P <0.05 in Dunnett's test, and “**” indicates P in Dunnett's test. <0.01 indicates that there is a significant difference.

In addition, human liver cancer cell line HepG2 was seeded at a cell density of 500 cells / mL in a 24-well low-adhesion plate (Corning) at 1 mL / well, and a final concentration of 0 or 10 μM EGCG was added, 20 ng / mL EGF, After culturing in serum-free DMEM medium containing 20 ng / mL bFGF, B27 (1:50 dilution, Invitrogen), 5 units / mL SOD, 200 units / mL catalase, the number of spheroids was measured (n = 3). ).

FIG. 31 (c) is a graph showing the results of the spheroid assay. In FIG. 31 (c), the result is expressed as an average value ± standard deviation, and “***” indicates that there is a significant difference at P <0.001 in Dunnett's test.

From the above results, it was revealed that the spheroid-forming ability of mouse melanoma cell line B16, mouse breast cancer cell line 4T1, and human liver cancer cell line HepG2 is reduced when a 67LR agonist is allowed to act. This result indicates that the 67LR agonist is a cancer stem cell inhibitor.

[Experimental example 31]
(Reduction of c-Met expression by 67LR agonist)
A 67LR agonist was added to the culture medium of mouse melanoma cell line B16 and human breast cancer cell line MDA-MB-231, and the effect on the expression of c-Met was examined. EGCG was used as the 67LR agonist.

Mouse melanoma cell line B16 was seeded at a cell density of 1 × 10 ⁴ cells / mL in a 24-well plate at 1 mL / well and pre-cultured in 5% FBS-DMEM medium for 24 hours. Thereafter, the cells were cultured for 96 hours in a medium (5 units / mL SOD, 200 units / mL catalase, 1% FBS) containing EGCG having a final concentration of 0, 1, 10 μM.

Subsequently, each cell was dissolved in a cell lysis buffer and collected, and the expression level of c-Met protein was measured by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 32A is a photograph showing the results of Western blotting and a graph in which the results are digitized. In the graph of FIG. 32 (a), the result is expressed as an average value ± standard deviation, “**” indicates that there is a significant difference at P <0.01 in Dunnett's test, and “***” indicates Dunnett. In this test, there is a significant difference at P <0.001.

In addition, human breast cancer cell line MDA-MB-231 was seeded at a cell density of 1 × 10 ⁴ cells / mL in a 24-well plate at 1 mL / well and pre-cultured in 10% FCS-RPMI medium for 24 hours. Thereafter, the cells were cultured for 96 hours in a medium (5 units / mL SOD, 200 units / mL catalase, 1% FCS) containing EGCG having a final concentration of 0, 1, 5, 10 μM.

Subsequently, each cell was dissolved in a cell lysis buffer and collected, and the expression level of c-Met protein was measured by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 32 (b) is a photograph showing the results of Western blotting and a graph in which the results are digitized. In the graph of FIG. 32 (b), the result is expressed as an average value ± standard deviation, and “***” indicates that there is a significant difference at P <0.001 in Dunnett's test.

From the above results, it was found that the expression level of c-Met in mouse melanoma cell line B16 and human breast cancer cell line MDA-MB-231 decreases when a 67LR agonist is allowed to act. This result indicates that the 67LR agonist is a c-Met expression inhibitor (expression suppressor).

[Experiment 32]
(Investigation of the effect of suppression of STARD3 expression on spheroid formation ability of breast cancer cells)
Spheroid-forming ability is one index of cancer stem cell function. Therefore, shRNA was used to suppress the expression of STARD3 in the human breast cancer cell line MDA-MB-231, and the influence on spheroid formation ability was examined.

MDA-MB-231 cells introduced with shRNA that suppresses the expression of STARD3 (STARD3-shRNA) and MDA-MB-231 cells introduced with control scrambled shRNA (Scr-shRNA) at a cell density of 2000 cells / mL, respectively. After seeding 1 mL / well on a 24-well low adhesion plate (Corning) and culturing for 21 days in a serum-free RPMI medium containing 20 ng / mL EGF, 20 ng / mL bFGF, B27 (1:50 dilution, Invitrogen) The number of spheroids was measured (n = 3).

FIG. 33 is a graph showing the results of the spheroid assay. In FIG. 33, the result is expressed as an average value ± standard deviation, and “***” indicates that there is a significant difference at P <0.001 in Student's t-test.

As a result, it was revealed that the suppression of STARD3 expression in the human breast cancer cell line MDA-MB-231 inhibits spheroid formation. That is, it became clear that suppression of STARD3 expression in breast cancer cells inhibits cancer stem cell function.

[Experimental Example 33]
(Investigation of the effect of suppression of STARD3 expression on c-Met signaling in breast cancer cells)
Using shRNA, the expression of STARD3 in human breast cancer cell line MDA-MB-231 was suppressed, and the influence on c-Met signaling was examined.

MDA-MB-231 cells introduced with shRNA that suppresses the expression of STARD3 (STARD3-shRNA) and MDA-MB-231 cells introduced with control scrambled shRNA (Scr-shRNA) were each 1 × 10 ⁶ cells / mL. The cells were seeded at a cell density of 1 mL / well in a 24-well plate and cultured in 10% FCS-RPMI medium for 24 hours.

Subsequently, each cell was lysed and collected in a cell lysis buffer, and phosphorylation of c-Met Tyr1234, expression of c-Met and expression of STARD3 were examined by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 34 (a) is a photograph showing the results of Western blotting.

FIG. 34 (b) is a graph in which the expression level of c-Met is quantified based on the result of FIG. 34 (a). FIG. 34 (c) is a graph in which the abundance of phosphorylated c-Met is quantified based on the result of FIG. 34 (a). In FIGS. 34 (b) and (c), the results are expressed as mean ± standard deviation, and “*” indicates that there is a significant difference at P <0.05 in Student's t-test.

As a result, it became clear that suppression of STARD3 expression in human breast cancer cell line MDA-MB-231 decreases the phosphorylation level of c-Met and the expression level of c-Met. This result indicates that suppression of STARD3 expression inhibits the c-Met pathway (c-Met signaling) in breast cancer cells.

[Experimental example 34]
(Investigation of the effect of suppression of STARD3 expression on spheroid formation ability of liver cancer cells)
Spheroid-forming ability is one index of cancer stem cell function. Thus, shRNA was used to suppress the expression of STARD3 in the human liver cancer cell line HepG2, and the influence on spheroid formation ability was examined.

24-well low-adhesion plates at a cell density of 200 cells / mL each of HepG2 cells introduced with shRNA that suppresses the expression of STARD3 (STARD3-shRNA) and HepG2 cells introduced with control scrambled shRNA (Scr-shRNA) (Corning) ) 1 ml / well, and cultured for 21 days in a serum-free DMEM medium containing 20 ng / mL EGF, 20 ng / mL bFGF, B27 (1:50 dilution, Invitrogen), and then the number of spheroids was measured (n = 4).

FIG. 35 is a graph showing the results of the spheroid assay. In FIG. 35, the results are expressed as mean values ± standard deviation, and “***” indicates that there is a significant difference at P <0.001 in Student's t-test.

As a result, it was revealed that the suppression of STARD3 expression in the human liver cancer cell line HepG2 inhibits spheroid formation. That is, it became clear that suppression of STARD3 expression in liver cancer cells inhibits cancer stem cell function.

[Experimental Example 35]
(Investigation of the effect of suppression of STARD3 expression on c-Met signaling in liver cancer cells)
shRNA was used to suppress the expression of STARD3 in the human liver cancer cell line HepG2, and the influence on c-Met signaling was examined.

2 mL of HepG2 cells introduced with shRNA that suppresses the expression of STARD3 (STARD3-shRNA) and HepG2 cells introduced with control scrambled shRNA (Scr-shRNA) at a cell density of 1 × 10 ⁵ cells / mL each in a 12-well plate Per well, and cultured in 10% FCS-DMEM medium for 48 hours.

Subsequently, each cell was lysed and collected in a cell lysis buffer, and phosphorylation of c-Met Tyr1234, expression of c-Met and expression of STARD3 were examined by Western blotting. Β-actin protein was measured as an internal standard (n = 3). FIG. 36 (a) is a photograph showing the results of Western blotting.

FIG. 36 (b) is a graph in which the abundance of phosphorylated c-Met is quantified based on the result of FIG. 36 (a). FIG. 36 (c) is a graph in which the expression level of c-Met is quantified based on the result of FIG. 36 (a). In FIGS. 34 (b) and (c), the results are expressed as mean values ± standard deviation, and “***” indicates that there is a significant difference at P <0.001 in Student's t-test.

As a result, it was found that suppression of STARD3 expression in the human liver cancer cell line HepG2 decreases the phosphorylation level of c-Met and the expression level of c-Met. This result indicates that suppression of STARD3 expression inhibits the c-Met pathway (c-Met signaling) in liver cancer cells.

According to the present invention, a new anticancer agent can be provided.

Claims (16)

1. Anti-cancer agent containing, as an active ingredient, an inhibitor of StAR-related lipid transfer domain containing 3 (STARD3) / cholesterol change / c-Met pathway.
2. The anticancer agent according to claim 1, wherein the STARD3 / cholesterol change / c-Met pathway inhibitor is a STARD3 inhibitor, a cholesterol kinetic regulator or a c-Met inhibitor.
3. The anticancer agent according to claim 1 or 2, wherein the inhibitor of STARD3 / change in cholesterol / c-Met pathway is a 67 kDa laminin receptor (67LR) agonist.
4. The anticancer agent according to any one of claims 1 to 3, wherein the 67LR agonist is epigallocatechin gallate or a derivative thereof.
5. The anticancer agent according to any one of claims 1 to 4, wherein the cancer is melanoma, breast cancer or liver cancer.
6. The anticancer agent according to claim 5, wherein the breast cancer is triple negative breast cancer.
7. The anticancer agent according to any one of claims 1 to 6, which is a cancer stem cell inhibitor.
8. The anticancer agent according to any one of claims 1 to 7, which is a cancer metastasis inhibitor.
9. A cancer therapeutic pharmaceutical composition comprising the anticancer agent according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
10. Measuring the expression level of STARD3 in a cell in the presence of a test substance;
    A step of determining that the test substance is an anticancer agent when the expression level of STARD3 is lower than the expression level in the absence of the test substance,
    Screening method for anticancer drug.
11. Measuring the activity of STARD3 in the presence of a test substance;
    Determining that the test substance is an anticancer agent when the activity of STARD3 is reduced compared to the activity in the absence of the test substance,
    Screening method for anticancer drug.
12. Measuring the amount of cholesterol present in the cells in the presence of the test substance;
    A step of determining that the test substance is an anticancer agent when the abundance of cholesterol is reduced as compared to the abundance in the absence of the test substance.
    Screening method for anticancer drug.
13. Measuring the expression level of c-Met in a cell in the presence of a test substance;
    a step of determining that the test substance is an anticancer agent when the expression level of c-Met is lower than the expression level in the absence of the test substance,
    Screening method for anticancer drug.
14. Measuring c-Met activity in the presence of a test substance;
    determining that the test substance is an anticancer agent when the activity of c-Met is reduced compared to the activity in the absence of the test substance,
    Screening method for anticancer drug.
15. The method for screening an anticancer agent according to any one of claims 10 to 14, wherein the cancer is melanoma, breast cancer or liver cancer.
16. The method for screening an anticancer agent according to claim 15, wherein the breast cancer is triple negative breast cancer.

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