KR101308216B1 - Composition for inhibiting cancer metastasis comprising CTHRC1 inhibitor and Method for screening cancer metastasis inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a CTHRC1 inhibitor as an active ingredient and a method for screening a cancer metastasis inhibitor. The composition of the present invention can be usefully used to suppress cancer metastasis. Moreover, according to the screening method of this invention, the therapeutic agent for inhibiting cancer metastasis can be identified efficiently.

Description

Composition for inhibiting cancer metastasis comprising CTHRC1 inhibitor and Method for screening cancer metastasis inhibitor}

The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising a CTHRC1 inhibitor as an active ingredient and a method for screening a cancer metastasis inhibitor.

Cancer (or tumor) is the product of abnormal, uncontrolled, disordered cell proliferation. In particular, it is classified as malignant if it has destructive growth, permeability and metastasis. The main cause of death in cancer patients is not due to early tumors, but to metastasis of cancer cells to other tissues. Accordingly, researches on the mechanism of cancer cell invasion and metastasis and a method of inhibiting the cancer cells are actively conducted. In the metastasis process of cancer cells, first, metastatic cancer cells are separated from the primary tumor, infiltrate into supporting structures such as epilepsy and basement membrane of normal tissue, attach to capillary walls of other target tissues, and decompose extracellular matrix and basement membrane. Outflow into the vessels, accompanied by angiogenesis in new tissue, secondary tumors are formed. Therefore, the metastasis process is composed of major steps such as migration, adhesion, and invasion, and if one of them can be prevented, cancer metastasis can be suppressed.

Invasion of cancer cells plays an important role in matrix degrading metalloproteinases (MMPs) secreted by attached cells. Matrix metalloproteinases (MMPs) are enzymes that degrade collagen and proteoglycans, including interstitial collagen enzymes, MMP2, and stromelsin, all of which are metal enzymes with Zn in the active center, and in the form of zymogens. It is secreted and activated by other proteolytic enzymes or organophosphorus compounds, and is inhibited by TIMP (tissue inhibitor of metalloproteinase) secreted with MMP, and is classified as MMPs because it has homology in cDNA sequence. In fact, cancer cells with active metastasis have higher activity of MMP2 or MMP9 than normal cells or non-metastatic cancer cells, and substances that inhibit them have been shown to inhibit cancer metastasis.

Since cancer metastasis is a major cause of death of cancer patients, there is an urgent need for development of effective anticancer drugs that can suppress cancer metastasis.

Under these backgrounds, the present inventors have studied the inhibition of cancer cell invasion and metastasis, and found that inhibition of CTHRC1 expression inhibits invasion of colorectal cancer cells, and cancer metastasis by CTHRC1 inhibits the It confirmed that it was by expression, and completed this invention.

It is an object of the present invention to provide a pharmaceutical composition for inhibiting cancer metastasis comprising an expression inhibitor of a CTHRC1 (collagen triple helix repeat containing 1) gene or an activity inhibitor of a CTHRC1 protein as an active ingredient.

Another object of the present invention is the step of (a) introducing a CTHRC1 (collagen triple helix repeat containing 1) gene into cancer cells; (b) measuring the expression level of phosphorylated extracellular signal-regulated kinases (ERK) in the cancer cells; (c) measuring the expression level of phosphorylated ERK after treating the cells with candidates; And (d) when the expression level of step (c) is lower than the expression level of step (b), determining the candidate substance as a cancer metastasis inhibitor.

In one embodiment, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising an expression inhibitor of a collagen triple helix repeat containing 1 (CTHRC1) gene or an activity inhibitor of a CTHRC1 protein as an active ingredient.

The present inventors confirmed that the infiltration of cancer cells significantly increased when the CTHRC1 gene was overexpressed, and the cancer metastasis caused by the CTHRC1 gene was found to be due to the increased expression of MMP9 through ERK activation.

According to one preferred embodiment, the expression inhibitor of the CTHRC1 gene may be an antisense oligonucleotide or siRNA specific to CTHRC1 RNA, and the activity inhibitor of the CTHRC1 protein may be an antibody specific for CTHRC1 protein.

As used herein, the term "metastasis" refers to the spread of cancer cells from one organ or part thereof to another organ or organ periphery, mainly having the ability to metastasize malignant cancer cells. Cancer cells escape from the primary cancer, penetrate into the lymphatic or vascular system, circulate through blood vessels, and grow in normal tissue in other parts of the body. Cancer metastasis is a typical feature of malignant cancers, the metastasis of which accounts for 90% of cancer deaths. In this context, infiltration and transition can be used interchangeably.

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA, or derivatives thereof, that contain oligonucleotide sequences that are complementary to the sequence of a particular mRNA, and bind to complementary sequences within the mRNA to bind the protein of the mRNA. It inhibits translation into. Antisense sequence for CTHRC1 refers to a DNA or RNA sequence that is complementary to CTHRC1 mRNA and capable of binding to CTHRC1 mRNA and is responsible for the translation, translocation, maturation, or any other overall biological function of CTHRC1 mRNA. May inhibit essential activity.

Antisense oligonucleotides can be synthesized in vitro and administered in vivo by conventional methods, or the antisense oligonucleotides can be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of synthesizing antisense RNA in vivo is to allow the antisense RNA to be transcribed using vectors of opposite recognition origin.

The design of antisense oligonucleotides that can be used in the present invention can be made with reference to the mRNA sequence of human CTHRC1 known in the art. For example, as described above, the sequence complementary to the CDS (coding sequence) of human CTHRC1 mRNA, the complementary sequence to the start codon and its surrounding sequence, the sequence complementary to the 5'-UTR, the complementary to the 3'-UTR The antisense oligonucleotide of the present invention can be designed with a typical sequence.

As used herein, the term "siRNA" refers to an oligonucleotide molecule capable of mediating RNA interference or gene silencing. Since siRNA can inhibit expression of a target gene, it can be provided as an efficient gene knockdown method or gene therapy method.

When the siRNA molecule is used in the present invention, the sense strand, that is, the sequence corresponding to the CTHRC1 mRNA sequence and the antisense strand, ie, the sequence complementary to the CTHRC1 mRNA sequence, may have a double stranded structure, or It can have a single chain structure with self-complementary sense and antisense strands.

siRNAs are not limited to the complete pairing of double-stranded RNA portions paired with RNA, but mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. It may include parts that are not paired by. The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the CTHRC1 gene can be inhibited by RNA interference (RNAi) effects.

Preferably, the siRNA may have a nucleotide sequence represented by SEQ ID NO: 13 and 14. In one embodiment of the present invention, when the siRNA consisting of polynucleotides represented by SEQ ID NO: 13 and 14 is introduced into the colon cancer cell line SW480 cells to inhibit the expression of the CTHRC1 gene, it was confirmed that the cancer cell invasion can be suppressed (See D in FIG. 6).

Antibodies that specifically bind to and inhibit activity of the CTHRC1 protein that may be used in the present invention may be polyclonal antibodies or monoclonal antibodies. Antibodies to the CTHRC1 protein can be prepared by methods commonly practiced in the art, such as fusion methods, recombinant DNA methods, or phage antibody library methods.

According to one preferred embodiment, the composition of the present invention can inhibit cancer metastasis by inhibiting the expression of Matrix metallopeptidase 9 (MMP9) through the inhibition of extracellular signal-regulated kinases (ERK) activity. In one embodiment of the present invention, as a result of experiments to confirm the mechanism of action of CTHRC1 on cancer metastasis, it was confirmed that the expression of MMP-9 is increased by the overexpression of CTHRC1, which is the expression of ERK by the overexpression of CTHRC1 It was confirmed that the activation (see Figure 6).

According to one preferred embodiment, the cancer may be colorectal cancer.

The composition of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The composition of the present invention is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of subject and its severity, age, sex, activity of the drug. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The route of administration of the composition according to the invention can be administered via any general route as long as it can reach the desired tissue, and as desired, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, Oral administration, intranasal administration, pulmonary administration, rectal administration, but is not limited thereto. In addition, a composition comprising an expression inhibitor of the CTHRC1 gene or an activity inhibitor of the CTHRC1 protein may be administered by any device that allows the active agent to migrate to the target cell.

In another aspect, the present invention provides a method for screening a cancer metastasis inhibitor. The screening method of the present invention,

(a) introducing a CTHRC1 (collagen triple helix repeat containing 1) gene into cancer cells;

(b) measuring the expression level of phosphorylated extracellular signal-regulated kinases (ERK) in the cancer cells;

(c) measuring the expression level of phosphorylated ERK after treating the cells with candidates; And

(d) if the expression level of step (c) is decreased from the expression level of step (b), the candidate material is included as a cancer metastasis inhibitor.

As used herein, the term "candidate" refers to a substance that will test for activity that inhibits cancer metastasis and includes any molecule, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide variety of compounds. It may include. Such candidates also include natural as well as synthetic materials.

In step (a), the introduction of the CTHRC1 gene into the cancer cell can be used without limitation, methods known in the art for introducing the gene into the cell, for example transforming the cell with an expression vector comprising a polynucleotide encoding the CTHRC1 protein. By infection, the CTHRC1 gene can be introduced into cancer cells.

According to one preferred embodiment, the cancer cells may be colon cancer cells, preferably SW480 cells which are human colon cancer cell lines.

In steps (b) and (c), measuring the expression level of phosphorylated ERK is to measure the degree of activation of ERK by overexpression of CTHRC1 gene introduced into cancer cells. Measurement of the expression level of phosphorylated ERK can be used without limitation methods known in the art for measuring the expression level of phosphorylated ERK, for example, expression level of phosphorylated ERK by Western blot using an antibody specific for phosphorylated ERK. Can be measured.

Step (d) is a step of judging a candidate substance that reduces the activity of ERK by overexpressed CTHRC1 as a cancer metastasis inhibitor. Candidates screened by the screening method of the present invention inhibit the activation of ERK by overexpression of CTHRC1, thereby suppressing the expression of MMP9, ultimately can inhibit cancer metastasis.

In one embodiment of the present invention, when the ERK inhibitor inhibits the activity of ERK, it was confirmed that the infiltration of cancer cells was significantly reduced (see Fig. 6 C).

As another aspect, the present invention can provide a composition for inhibiting cancer metastasis comprising a cancer metastasis inhibitor screened by the screening method.

According to the present invention, CTHRC1 upregulates expression of MMP9 through activation of ERK. Therefore, the composition of the present invention that inhibits the expression or activity of CTHRC1 can be usefully used to inhibit cancer metastasis.

In addition, according to the screening method of the present invention, cancer metastasis inhibitors can be screened efficiently.

1 is a graph showing cell infiltration activity by CTHRC1. Infiltration of SW480-Mock cells and SW480-CTHRC1 cells was assessed with a 24-well modified Boyden chamber.
2 shows RT-PCR results showing the transcription level of EMT markers in SW480-Mock cells and SW480-CTHRC1 cells.
Figure 3 shows the Western blot results showing the protein expression level of the EMT markers in SW480-Mock cells and SW480-CTHRC1 cells.
4 shows the correlation between CTHRC1 and MMP9 in tumor tissues. In order to test whether the expression of CTHRC1 and MMP9 correlated in colorectal cancer tissues, transcripts obtained from colorectal cancer tissues were analyzed by RT-PCR (A) and quantitative PCR (B).
Figure 5 shows the results of measuring the expression level of MMP9 by RT-PCR after treatment with various inhibitors in SW480-CTHRC1 cells.
6 shows that CTHRC1 can promote infiltration of SW480 cells through activation of the MEK-ERK signaling pathway. (A) Influence of U0126 on MMP9 and phosphorylated ERK. SW480-CTHRC1 cells were treated with DMSO or 10 μM U0126 for 24 hours. (B) Luciferase reporter assay results are shown. Transcriptional activation of Elk1 promoter by ERK activation signaling was measured in SW480 cells using Dual-Luciferase Assay. (C) The result of measurement of cell invasion by overexpression of CTHRC1 in the presence of an ERK inhibitor (10 μM U0126) is shown. (D) SW480-Mock, SW480-CTHRC1 and CTHRC1 show the invasion capacity of knocked down SW840 cells.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Experimental Method

1-1. CHTRC1 Cell line expression

Human colon cancer cell lines, 1% penicillin / streptomycin (Lonza) and open the-addition of fire, activate 10% FBS (heat-inactivated fetal bovine serum) were cultured in a DMEM culture medium, the culture is 5% CO ₂ and 37 ℃ The temperature was advanced in the humidification chamber. Human CTHRC1 cDNA was PCR amplified using 5'-GATATCATGCGACCCCAGGGC-3 '(SEQ ID NO: 1) and 5'-CTCGAG TTAATGGTGATGGTGATGATGTTTTGGTAGTTCTTCAATAATGATGC-3' (SEQ ID NO: 2) primers. The amplified PCR product was cloned into a His-tagged expression vector, pcDNA3.1 / hygro (+) (Invitrogen) vector. SW480 cells were transfected using Lipofectamine ^™ 2000 (Invitrogen) as the expression vector. After 24 hours of culture, 0.2 mg / mL hygromycin B (calbiochem) was treated to select the transfected cells.

1-2. RT - PCR

Total RNA was extracted from the cultured cells using total RNA extraction solution (RiboEx ^™ , GeneAll, Korea). RT-PCR was performed using the Verso 1-step RT-PCR kit (Thermo Scientific, Abgene). The sequence of the used primer is as follows.

CTHRC1: 5'-TGGACACCCAACTACAAGCA-3 '(SEQ ID NO: 3) and 5'-GAACAA GTGCCAACCCAGAT-3' (SEQ ID NO: 4);

MMP9: 5'-GATGCGTGGAGAGTCAAAT-3 '(SEQ ID NO: 5) and 5'-CACCAAACTGGATGACGATG-3' (SEQ ID NO: 6);

E-cadherin: 5'-TGATTCTGCTGCTCTTGCTG-3 '(SEQ ID NO: 7) and 5'-CGAGTCCCCTAGTCGTCCT-3' (SEQ ID NO: 8);

Cox2: 5'-TTCAAATGAGATTGTGGAAAAATTGCT-3 '(SEQ ID NO: 9) and 5'-AGATCATCTCTGCCTGAGTATCTT-3' (SEQ ID NO: 10); And

GAPDH: 5'-CCATCACCATCTTCCAGGAG-3 '(SEQ ID NO: 11) and 5'-ACAGTCTTCTGGGTGGCAGT-3' (SEQ ID NO: 12)

1-3. Western Blat

The cells were lysed with protein extracts (PRO-PREP ^™ , iNtRON, Korea) on ice. Cell lysates were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Thereafter, the polyvinylidene difluoride membrane was incubated at room temperature for 1 hour in a TBST solution (10 mmol / L Tris-HCl, pH 8.0), 150 mmol / L NaCl, and 0.05% Tween 20 added with 5% skim milk powder. The primary antibody was treated at 4 ° C. for 1 day. Binding antibodies were visualized as secondary antibodies bound to horseradish peraxidase using enhanced chemiluminescence reagents (Millipore). Antibodies used include anti-E-cadherin Ab (BD pharmingen), anti-Vimentin Ab, anti-Cox2 Ab, anti-phospho-ERK1 / 2 Ab, anti-ERK1 / 2 antibody (Cell signaling), anti-His Ab (IG Therapy), anti-α-tubulin Ab, anti-Mouse IgG peroxidase conjugate, anti-Rabbit IgG peroxidase conjugate (Sigma).

1-4. siRNA  Experiment

Synthetic siRNA duplex oligomers were designed to inhibit CTHRC1 expression. The sequences of the siRNA oligo duplex (siCTHRC1) are 5'-CCCAUUGAAGCUAUAAUUU-3 '(SEQ ID NO: 13) and 5'-AAAUUAUAGCUUCAAUGGG-3' (SEQ ID NO: 14). Transient transfection of siRNA oligo duplexes was performed using G-fectin (Genolution pharmaceuticals, Inc). After 48 hours of incubation, the cells were lysed and the efficiency of siRNA oligo duplexes was confirmed by Western blot.

1-5. Cell Infiltration Essay

In vitro cell invasion assays were performed using a modified Boyden chamber assay. Cells were incubated to ˜70% -80% growth and then serum deficient for 24 hours. After separating cells with trypsin / EDTA, washing with DMEM containing 1% FBS and resuspending in serum-free medium, 300 μl of cells (5 × 10 ⁵ cells ml ⁻¹ ) were added to the QCMTM 24 well cell infiltration assay kit ( Inoculated into the upper chamber of Millipore). Serum containing medium (10%) was used as chemoattractant in the lower chamber. After 24 hours of incubation, the cells infiltrated into the bottom surface of the collagen coated membrane were analyzed according to the manufacturer's instructions.

1-6. gelatin Zymography  ( Gelatin zymography )

Cells were seeded in 10 cm culture plates at a density of 6 × 10 ⁶ cells. After 16 hours the cells were washed with PBS and incubated in serum free medium for 72 hours. The conditioned medium was concentrated and recovered by centrifugation. Proteins were uniformly mixed with non-reducing Laemml's sample buffer and electrophoresed on 10% SDS-PAGE gels containing 0.1% (w / v) gelatin. The gel was then washed for 30 minutes in Zymogram renaturing buffer (2.5% Trypton X-100), Zymogram developing buffer, 50 mM Tris-HCl (pH 7.5), 200 mM NaCl 5 mM CaCl ₂ , 5 μM ZnCl ₂ ) was incubated at a temperature of 37 ° C. for 24 hours. Gelatin soluble activity of MMP9 was detected by Coomassie blue staining as a clear band on the gel.

1-7. Luciferase  Reporter Essay

Cells were grown to account for about 60% of the total area of a 24-well plate, and cells were transfected with ElK1 luciferase reporter gene construct (Panomics) and pRL-TK together using Lipofectamine ^™ 2000 (Invitrogen). After obtaining cell extracts 24 hours after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). All experiments were repeated three times and three times.

Example  2: Invasion of colorectal cancer cells CTHRC1 The impact of

In order to confirm the function of CTHRC1 as a tumor gene candidate gene of colorectal cancer, the invasion activity of SW480 cell line (SW480-CTHRC1) overexpressed CTHRC1 was investigated. As a result, the infiltration of SW480-CTHRC1 cell line increased about 2 times compared to Mock control cells (FIG. 1).

In order to find genes regulated by overexpressed CTHRC1 status, RT-PCR was performed to detect epithelial-mesenchymal transtion (EMT) -related genes. As a result, MMP9 and Cox2 transcripts were increased in SW480-CTHRC1 cell line, and E-cadherin was slightly decreased (FIG. 2).

Protein levels of E-Kedherin and vimentin were confirmed by immunoblotting and MMP9 was detected by zymography. As a result, E-Kedherin, an inhibitory protein of EMT, was decreased, and protein levels of non-mentin and MMP9, EMT-related proteins, were increased. In particular, expression of MMP9 was very sensitive to CTHRC1 at both RNA and protein levels (FIG. 3).

To confirm the relationship between CTHRC1 and MMP9, RT-PCR and real-time PCR were performed on transcripts from the same patient. As a result, expression of CTHRC1 was detected in most colorectal cancer patients and CTHRC1 positive tissues expressing MMP9 (FIG. 4). These results indicate that CTHRC1 is a positive regulator of MMP9 expression that induces tumor invasion.

Example  3: CTHRC1 of ERK  Active MMP9 Confirm upward adjustment of

To identify how CTHRC1 regulates the expression of MMP9, several kinase inhibitors (phospholipase C inhibitors, MEK1 / 2 inhibitors, JNK inhibitors, p38 MAPK inhibitors, NFkB inhibitors and Rac1 inhibitors) were treated. As a result, expression of MMP9 was significantly suppressed in SW480-CTHRC1 cell line, especially when treated with MEK 1/2 inhibitor (U0126) (FIG. 5). The SW480-CTHRC1 cell line showed higher ERK activity than the Mock control, and the MEK inhibitor was effective in reducing ERK activity as well as expression of MMP9 (FIG. 6A).

To confirm the activation of the ERK pathway, an ELK1 luciferase assay was performed that is directly regulated by activated ERK. As a result, the SW480-CTHRC1 cell line showed high ELK1 luciferase activity, which was inhibited by MEK inhibitors (FIG. 6B). Infiltration by CTHRC1 was also inhibited by ERK inhibition (FIG. 6C). Increased infiltration and ERK activity was reduced by CTHRC1 knockdown in SW480-CTHRC1 cell line (FIG. 6D). These results indicate that CTHRC1 can induce high invasion of colorectal cancer cells through upregulation of ERK mediated MMP9.

Attach an electronic file to a sequence list

Claims (7)

CTHRC1 (collagen triple helix repeat containing 1) Gene expression inhibitor or a pharmaceutical composition for inhibiting cancer metastasis comprising an activity inhibitor of CTHRC1 protein as an active ingredient.
The composition of claim 1, wherein the expression inhibitor of the gene is an antisense oligonucleotide or siRNA specific for CTHRC1 RNA, and the activity inhibitor of the protein is an antibody specific for CTHRC1 protein.
The composition of claim 2, wherein the siRNA has a nucleotide sequence of SEQ ID NOs: 13 and 14.
The composition of claim 1, wherein the composition inhibits the expression of Matrix metallopeptidase 9 (MMP9) through the inhibition of extracellular signal-regulated kinases (ERKs).
The composition of claim 1, wherein the cancer is colorectal cancer.
(a) introducing a CTHRC1 (collagen triple helix repeat containing 1) gene into cancer cells;
(b) measuring the expression level of phosphorylated extracellular signal-regulated kinases (ERK) in the cancer cells;
(c) measuring the expression level of phosphorylated ERK after treating the cells with candidates; And
(d) when the expression level of step (c) is lower than the expression level of step (b), determining the candidate substance as a cancer metastasis inhibitor.
The method of claim 6, wherein the cancer cells are colorectal cancer cells.

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