PHARMACEUTICAL COMPOSITION FOR PREVENTING AND TREATING CANCER OR CANCER METASTASIS, CONTAINING FSTL1 PROTEIN AS ACTIVE INGREDIENT

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating cancer, cancer metastasis and bone metabolic diseases, which comprises FSTL1 (Follistatin-like 1) protein as an active ingredient. More specifically, FSTL1 inhibits AKT activity and increases activity of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome 10), while inhibiting DNA binding of NF-B (nuclear factor-B) and blocking the expression of target genes IL-6, GM-CSF, MMP-9, VEGF and ICAM1, and exhibits significant inhibitory effects on breast cancer growth and metastasis in animal models. Therefore, it can be useful for inhibiting cancer metastasis including breast cancer.

Claims

1. A method for treating or preventing cancer or cancer metastasis, comprising administering a pharmaceutically acceptable amount of a FSTL1 protein, a vector comprising a polynucleotide encoding said protein, or a cell comprising said vector or culture fluid thereof to a subject having the cancer.

2. (canceled)

3. The pharmaceutical composition according to claim 1, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer.

4. The method according to claim 1, wherein the vector said is linear DNA, plasmid DNA or recombinant viral vector.

5. The method according to claim 4, wherein the recombinant virus is any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, Herpes simplex virus and Lentivirus.

6. The method according to claim 1, wherein the cell is any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells.

7. The method according to claim 1, wherein the cancer metastasis is breast cancer bone metastasis.

8. A method for treating or preventing a bone metabolic disease comprising administering a pharmaceutically acceptable amount of a FSTL1 protein, a vector comprising a polynucleotide encoding said protein, a cell comprising said vector, or culture fluid thereof to a subject suffering from a bone metabolic disease.

9. (canceled)

10. The method according to claim 8, wherein the bone metabolic disease is at least one selected from the group consisting of bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumors, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease and Paget's disease.

11. (canceled)

12. A screening method of agent for preventing or treating cancer or cancer metastasis comprises: 1) treating the test substance with a cancer cell line as an experimental group; 2) measuring the expression level of FSTL1 (Follistatin-like 1) protein in the treated cell line of step 1); and 3) selecting the test substance whose FSTL1 protein expression in step 2) is increased compared to the control.

13. The method according to claim 12, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer.

14. The method according to claim 12, wherein the cancer metastasis is breast cancer bone metastasis.

15. The method according to claim 12, wherein the cancer cells of step 1) express AP-1 (Activator protein-1).

16. The method according to claim 12, wherein the FSTL1 protein of step 2) inhibits AKT activity and increases the activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10).

17. The method according to claim 12, wherein the FSTL1 protein of step 2) blocks the signal pathway of NF-kB.

18. The method according to claim 12, wherein the degree of expression of the FSTL1 protein in step 2) is determined by any one selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting and flow cytometry (FACS).

19-26. (canceled)

Description

BRIEF DESCRIPTION OF FIGURES

[0029] FIG. 1 shows the expression of FSTL1 (Follistatin-like 1) in the MDA-MB-231 breast cancer cell line (MDA/T) transduced a c-jun/AP-1 mutant (Dominant-Negative Mutant) gene by using retrovirus. [0030] MDA/V: control, MDA-MB-231 breast cancer cell line transduced with retrovirus: protein F: FSTL1 protein.

[0031] FIG. 2 is a graph showing inhibition of AKT activity by FSTL1 protein and induction of tumor suppression function of PTEN (phosphatase and tensin homolog deleted on chromosome 10).

[0032] FIG. 3 is a graph showing inhibition of DNA binding and transcriptional regulation of NF-B by FSTL1 protein and blocking of NF-B target protein expression.

[0033] FIG. 4 shows inhibition of adhesion and invasion mechanism of breast cancer cells by FSTL1 protein and blocking effect of tumor angiogenic factor expression in vitro models.

[0034] FIG. 5 is a graph showing the effect of FSTL1 protein on breast tumor growth, bone metastasis inhibition, and osteoclast-reducing effects in animal models.

[0035] FIG. 6 is a graph showing the inhibitory effect of FSTL1 protein on osteoclastogenesis in an ex vivo model of mouse bone marrow cells.

DESCRIPTION OF EMBODIMENTS

[0036] Hereinafter, the present invention is described in more detail.

[0037] The present invention provides a pharmaceutical composition for preventing and treating cancer comprising FSTL1 (Follistatin-like 1) protein as an active ingredient.

[0038] The present invention also provides a pharmaceutical composition for preventing and treating cancer comprising a vector comprising a polynucleotide encoding an FSTL1 protein, a cell containing the vector, or culture thereof as an active ingredient.

[0039] The cancer is preferably one or more selected from the group consisting of melanoma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, thyroid tumor, gastric cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, glioblastoma, endometrial cancer, renal cancer, colon cancer, pancreatic cancer, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, cholangiocarcinoma, small intestine cancer, pediatric malignant cancer and epidermal cancer, more preferably one or more selected from the group specifically consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, and renal cancer, and more particularly, breast cancer is more preferable a.

[0040] The vector may be a linear DNA expressed in human or animal cells, a plasmid vector, a vector containing a viral expression vector or a recombinant retrovirus vector, a recombinant adenovirus vector, and recombinant virus vectors comprising a recombinant adeno-associated virus vector (AAV), a recombinant Herpes simplex virus vector or a recombinant Lentivirus vector, but is not limited thereto.

[0041] The cells above stated are preferably selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells and established tumor cells, and both the cell itself and the culture medium in which the cells are cultured can be used.

[0042] In the specific experimental examples of the present invention, the inventors identified the target protein of the activator protein-1 (AP-1) known to be related with the cancer growth and metastasis, and as a result, the expression of FSTL1 protein was confirmed in the MDA-MB-231 breast cancer cell line transduced with retrovirus expressing the dominant negative mutation of c-jun/AP-1 (see FIG. 1).

[0043] In addition, as a result of identifying the signal pathway of FSTL1 anticancer activity of, confirmed that FSTL1 of the present invention inhibits phosphorylation of AKT1 in breast cancer and metastatic prostate cancer cell lines (see FIGS. 2a and 2d), which indicates that FSTL1 is an excellent inhibitor of AKT activity by inhibiting the activity significantly versus a compound known as PI-3 kinase inhibitor, an upstream signal target protein of AKT (see FIG. 2c). FSTL1 increases the tumor suppression effect by inhibiting the phosphorylation of tumor suppressor PTEN, and the effect increases following to the amount thereof increases (see FIG. 2b).

[0044] Further, as a result of confirming the regulation effect of inhibiting NF-B DNA binding in breast cancer cells, confirmed that FSTL1 can inhibit the expression of NF-B transcriptional gene, thereby it can act as an inhibitor of breast cancer metastasis (see FIG. 3).

[0045] Also, confirmed that FSTL1 significantly inhibits the expression of the adhesion and invasion mechanism mediated molecule of breast cancer cells, and tumor angiogenesis factors those regulated by NF-B in vitro models (see FIG. 4a), and the invasion ability remarkably reduced by about 79% or more (see FIG. 4b).

[0046] In addition, as a result of confirming the effect of FSTL1 in animal models, confirmed that FSTL1 significantly inhibits tumor growth and breast cancer bone metastasis (see FIG. 5), as well as the quantitative reduction of osteoclasts (see FIG. 5 bottom right) and inhibition of osteoclasts generation in ex vivo models (see FIG. 6).

[0047] Therefore, FSTL1, AP-1 target gene of the present invention inhibits AKT activity being associated with tumorigenesis and cell viability, increases the function of PTEN, tumor suppressor protein, and inhibits the expression of target protein by interfering the signal pathway of NF-B, and thus it can be useful in the prevention and treatment of cancer since it represents a significant growth inhibition of breast cancer and bone metastasis inhibitory effect in animal models.

[0048] The therapeutically effective amount of the composition of the present invention may vary depending on the variety of factors, such as the method of administration, the site of the subject, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations for determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E. W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0049] The present composition may also include carriers, diluents, excipients, or a combination of two or more thereof, which are commonly used in biological products. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., a buffered saline solution, sterilized water, Ringer's solution, a buffer solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and if necessary, other conventional additives such as an antioxidant, a buffer, bacteriostatic agent, may be added thereto. In addition, a diluent, a dispersing agent, a surfactant, a binder and a lubricant may be additionally added and formulated to a main use formulations such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule or a tablet. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton Pa., 18th, 1990), in a suitable manner in the art.

[0050] The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the protein, based on the total weight of the composition.

[0051] The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the intended method, and the dosage varies on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, more preferably once to several times a day.

[0052] The vector containing the polynucleotide encoding the FSTL1 protein of the present invention preferably contains 0.05 to 500 mg, more preferably 0.1 to 300 mg, and in case of recombinant virus comprising the polynucleotide encoding the FSTL1, it is preferable to contain 10.sup.3-10.sup.12 IU (10-10.sup.10 PFU, more preferably 10.sup.5-10.sup.10 IU, but is not limited thereto.

[0053] In addition, in the case of a cell comprising a polynucleotide encoding the FSTL1 protein of the present invention, it is preferable to contain 10.sup.3-10.sup.8, more preferably 10.sup.4-10.sup.7, but is not limited thereto.

[0054] In addition, the effective dose of composition of the present invention containing a vector including a polynucleotide encoding the FSTL1 protein or cells as an active ingredient is 0.05 to 12.5 mg per 1 kg of body weight in the case of containing a vector, 10.sup.7 to 10.sup.11 virus particles (10.sup.5 to 10.sup.9 IU) per 1 kg of body weight in case of containing recombinant virus, 10.sup.3 to 10.sup.6 cells per 1 kg of body weight in case of cells, preferably 0.1 to 10 mg/kg in case of vector, 10.sup.8 to 10.sup.10 particles (10.sup.6 to 10.sup.8 IU)/kg in case of recombinant virus, 10.sup.2 to 10.sup.5 cells/kg in case of cells, and can be administered 2 to 3 times a day. Above stated compositions are not necessarily limited to them, and may vary depending on the condition of the patient and the degree of disease.

[0055] The present invention also provides a health food for prevention and improvement of cancers, which comprises an FSTL1 protein as an active ingredient.

[0056] FSTL1, AP-1 target gene of the present invention, inhibits AKT activity being associated with tumorigenesis and cell viability, increases the function of PTEN, tumor suppressor protein, and inhibits the expression of target protein by interfering the signal pathway of NF-B, and thus it can be effectively used as an effective ingredient of health food for cancer prevention and improvement since it represents significant efficacy on growth inhibition of breast cancer and bone metastasis inhibitory activities in animal models.

[0057] The present invention also provides a pharmaceutical composition for inhibiting cancer metastasis comprising FSTL1 protein as an active ingredient.

[0058] The present invention also provides a pharmaceutical composition comprising a vector including a polynucleotide encoding an FSTL1 protein, a cell comprising the vector, or culture fluid thereof as an active ingredient for inhibiting cancer metastasis.

[0059] It also provides health food comprising a FSTL1 protein, a vector comprising a polynucleotide encoding the FSTL1 protein, a cell containing the vector, or culture fluid thereof as an active ingredient for improving cancer metastasis.

[0060] The cancer is preferably one or more selected from the group consisting of melanoma, small cell lung cancer, non-small cell lung cancer, glioma, liver cancer, thyroid tumor, gastric cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, glioblastoma, endometrial cancer, renal cancer, colon cancer, pancreatic cancer, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, cholangiocarcinoma, small intestine cancer, pediatric malignant cancer and epidermal cancer, more preferably one or more selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, and renal cancer, and more particularly, it is more preferably a breast cancer, but is not limited thereto.

[0061] In the specific experimental examples of the present invention, confirmed that FSTL1 of the present invention significantly inhibits the expression of MMP-9, VEGF and ICAM-1, molecules being related with cancer metastasis (see Table 1 and FIG. 4).

[0062] Therefore, according to the present invention, FSTL1, a target gene of AP-1 (Activator protein 1), inhibits AKT activities being associated with tumorigenesis, cell proliferation and cell viability; increases the activity of PTEN, tumor suppressor agent; inhibit NF-B signal pathway; blocks the expression of the target genes IL-6, GM-CSF, MMP-9, VEGF and ICAM1, target gene of NF-B, and exhibits significant inhibition of breast cancer growth and inhibition of bone metastasis in animal models, and thus it can be used as an active ingredient of composition for inhibiting cancer metastasis.

[0063] The present invention also provides a pharmaceutical composition comprising as an active ingredient a FSTL1 protein, a vector comprising a polynucleotide encoding the FSTL1 protein, a cell containing the vector, or culture fluid thereof for the prevention and treatment of bone metabolic diseases.

[0064] The present invention also provides health food comprising a FSTL1 protein, a vector comprising a polynucleotide encoding the FSTL1 protein, a cell containing the vector, or culture fluid thereof as an active ingredient for the prevention and improvement of bone metabolic diseases.

[0065] The bone metabolic diseases include preferably any one selected from the group consisting of bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid cancer metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumors, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease and Paget's disease, most preferably a breast cancer bone metastasis.

[0066] FSTL1 according to the present invention, a target gene of AP-1, inhibits AKT activity being associated with tumorigenesis, cell proliferation and cell viability; increases the activity of PTEN, a tumor suppressor agent; inhibits NF-B signal pathway; blocks the expression of the target genes IL-6, GM-CSF, MMP-9, VEGF and ICAM1, target gene of NF-B, and exhibits significant inhibition of breast cancer growth and inhibition of bone metastasis in animal models, and thus it can be used as an active ingredient of composition for preventing and treating bone metabolic diseases including breast cancer bone metastasis.

[0067] In addition, the present invention provides a screening method of agent for preventing or treating cancer or cancer metastasis comprises:

[0068] 1) treating the test substance with a cancer cell line as an experimental group;

[0069] 2) measuring the expression level of FSTL1 (Follistatin-like 1) protein in the treated cell line of step 1); and

[0070] 3) selecting the test substance whose FSTL1 protein expression in step 2) is increased compared to the control.

[0071] Preferably, the cancer is one or more selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer, and the cancer metastasis is preferably breast cancer bone metastasis, but is not limited thereto.

[0072] Preferably, the cancer cell of step 1) expresses AP-1 (Activator protein-1), and the FSTL1 protein of step 2) inhibits AKT activity, activates PTEN (phosphatase and tensin homolog deleted on chromosome 10), and interferes the signal pathway of NF-kB.

[0073] The expression level of the FSTL1 protein in the step 2) can be measured by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting and flow cytometry (FACS), but is not limited thereto.

[0074] FSTL1 according to the present invention, a target gene of AP-1, inhibits AKT activity being associated with tumorigenesis, cell proliferation and cell viability; increases the activity of PTEN a tumor suppressor agent, inhibits NF-B signal pathway; blocks the expression of the target genes IL-6, GM-CSF, MMP-9, VEGF and ICAM1, target gene of NF-B, and exhibits significant inhibition of breast cancer growth and inhibition of bone metastasis in animal models. Therefore, a substance that increases the expression of FSTL1 in cancer cells can be used to a therapeutic agent or therapeutic supplements for cancer or cancer metastasis.

EXAMPLES

[0075] Hereinafter, the present invention is described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

<Example 1> Culture of Breast Cancer Cell Lines and Prostate Cancer Cell Lines

[0076] Human breast cancer cells (MDA-MB-231 cells) and prostate cancer cells (PC-3M cells) used in the present invention were purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA). Cells were cultured in a medium including Dulbecco's modified Eagle's medium/Nutrient Mixture Ham's F12 (DMEM/F12, Gibco/BRL, Gaithersburg, Md., USA), 10% FBS, 100 units/ml penicillin, 100 g/mL Streptomycin (Gibco/BRL). When cell number (i.e., cell density) reaches 80-90% of a 10-cm culture dish, it is washed twice with phosphate buffered saline (PBS) and treated with Trypsin-EDTA (Gibco/BRL). Alternatively, samples were prepared for in vivo or in vitro experiments, and cell culture medium was changed every 2 to 3 days.

<Example 2> Identification of Target Protein of Transcription Factor AP-1 (Activator Protein-1)

[0077] In order to analyze the pattern of intracellular gene expression by the transcription factors AP-1 and FSTL1 known to be related to the growth and metastasis of cancers, the dominant-negative mutant gene of c-jun/AP-1 or the FSTL1 clone was analyzed in a Gene pool showing increased expression or decreased expression respectively in each MDA-MB-231 breast cancer cell line transduced with retrovirus by Northern blotting and cDNA microarray analysis (i.e., Human genome-U133 plus 2.0 genechip, Affymetrix, Inc.).

[0078] As a result, as shown in FIG. 1, confirmed that the FSTL1 gene was up-expressed by the introduction of the dominant negative mutation of c-jun/AP-1 and the FSTL1 gene and protein expression was inhibited in the control breast cancer cell line (FIG. 1).

[0079] Therefore, confirmed that FSTL1 protein is correlated with tumor suppression.

<Experimental Example 1> Identification of FSTL1 Antitumor Activity Signal Pathway

[0080] The FSTL1 condition medium (5%, v/v) and 100 ng/ml of IGF-1 (insulin-like factor-1) were treated to the breast cancer cell lines (MDA-MB-231 cell), prostate cancer cell lines (PC-3M) cultured in Example 1, or a MDA-MB0231 breast cancer cell lines transfected with retrovirus as a control, and then assessed Akt 1 and PTEN (phosphatase and tensin homolog deleted on chromosome 10) activities by using Western blotting. On the other hand, PI-3K (phosphatidylinositol-3 kinase) inhibitor or Akt inhibitor LY294002 was used as a control.

[0081] As a result, as shown in FIG. 2, the FSTL1 condition medium of the present invention inhibited the phosphorylation of Akt1 (FIGS. 2a and 2d) and inhibited phosphorylation of the cancer inhibitor PTEN, an upstream signal target of PI-3K, and increased tumor suppression effect, and specifically the FSTL1 condition medium of the present invention significantly inhibited the phosphorylation of Akt1 than the compound known as the conventional PI-3K inhibitor or Akt inhibitor (FIG. 2b).

<Experimental Example 2> Identification of NF-B Signal Pathway Regulation of FSTL1

[0082] In order to confirm the NF-B signal pathway of FSTL1, genes down-regulated by FSTL1 were analyzed using Human Genome-U133 Plus 2.0 Genechip and Affymetrix.

[0083] As a result, as shown in Table 1, it was confirmed that the gene down-regulated by FSTL1 is a gene having functions such as angiogenesis, cell adhesion/migration, cell growth, inflammation and tumor formation regulation and the like (Table 1).

TABLE-US-00001 TABLE 1 Fold Function Gene ID Gene Symbol, Description Change Angiogenesis AF043337.1 IL8, Interleukin 8 13.76 NM_000600.1 IL6, Interleukin 6 (interferon, 4.21 beta 2) S69738.1 CCL2, Monocyte chemotactic 18.35 protein 1 Cell Adhesion NM_002207.1 ITGA9, Integrin, alpha 9 15.00 Cell Growth NM_001511.1 CXCL1, GRO1 oncogene 10.78 Development NM_006576.1 AVIL, Advillin 2.47 Inflammation NM_000064.1 C3, Complement component 3 34.58 M57731.1 CXCL2, GRO-beta 6.49 NM_002090.1 CXCL3, GRO3 oncogene 7.77 Stimulation M11734.1 CSF2, GM-CSF 63.11 NM_000759.1 CSF3, G-CSF 17.14 Tumorigenesis NM_002387.1 MCC, mutated in colorectal 3.08 cancers AI829726 CCND1, Cyclin D1 4.09 Human Genome-U133 Plus 2.0 Gene Chip, Affymetrix, Inc.

[0084] At the mRNA level, FSTL1 also blocked the expression of IL-8 (interleukin-8) cytokines regulated by NF-B, and a granulocyte macrophage colony stimulating factor (GM-CSF)(FIG. 3a).

[0085] In addition, it was confirmed that FSTL1 quantitatively inhibits the binding of NF-kB with cytokine promoter through Luciferase reporter analysis (FIG. 3b).

[0086] Furthermore, it was confirmed that FSTL1 interferes the transcriptional activity of NF-B through an electromobility gel shift assay (EMSA) (FIG. 3c). However, it was confirmed that FSTL1 does not follow the TNF- dependent signal pathway. Following to the FSTL1 treatment, IB was not only degraded, but also damaged the phosphorylation of Akt and IB.

[0087] Therefore, it was confirmed that FSTL1 can regulate the induction of Akt-mediated NF-B signaling and play a role as an inhibitor of breast cancer metastasis.

<Experimental Example 3> Confirmation of Inhibitory Effect of FSTL1 on NF-B Signal Pathway

[0088] Expression of various FSTL1-mediated molecules (MMP-9, VEGF, ICAM-1) was analyzed. These molecules are regulated by NF-B and are involved in metastasis-related invasion, angiogenesis and cell adhesion. In particular, MMP-9 has been implicated in tumor angiogenesis and metastasis as well as activation of growth factor required for cancer which stimulates breast cancer activity. In addition, MMP-9 is expressed by osteoclasts and is required for osteoclast migration. Therefore, MMP-9 plays an important role in the osteoclast microenvironment being associated with bone resorption.

[0089] The expression of MMP-9, VEGF, and ICAM-1 was analyzed by immunoblot and zymography using the breast cancer cell lines expressing FSTL1 and control breast cancer cell lines prepared in Example 2 above.

[0090] As a result, as shown in FIG. 4, it was confirmed that FSTL1 of the present invention significantly inhibited all of the factors in vitro (FIG. 4a) and remarkably decreased FSTL1-mediated invasion ability by about 79% or more (FIG. 4b).

<Experimental Example 4> Confirmation of Effect of FSTL1 in Animal Model

[0091] <4-1> Preparation of Experimental Animals

[0092] Nude mice were BALB/c/nu (Korea Research Institute of Bioscience and Biotechnology, Korea) as a basic species, and all 20 females were 68 weeks old and weighing about 20 g. Sterilized water and feed were supplied in sterilized cage, and a clean environment was maintained by placing the lamina flow in a continuously held clean bench, and all procedures were also carried out in clean bench. For the day and night biological cycle of nude mice, the interior lights were illuminated for 12 hours and blinked for 12 hours.

[0093] <4-2> Identification of Tumor Growth Inhibitory Effect

[0094] The breast cancer cell line expressing FSTL1 prepared in Example 2 above was inoculated subcutaneously into a nude mouse using a 1 cc syringe and a 25 gauge needle to prepare a nude mouse prepared in Experimental Example <4-1>, and then nude mice transplanted with breast cancer cell lines and tumor volume were measured. Tumor volume was measured by caliper length and width once every 3 days, and the tumor volume was calculated by the formula of Gutman et al.

[0095] As a result, as shown in FIG. 5a, it was confirmed that the mice transplanted with the breast cancer cell line expressing FSTL1 significantly inhibited tumor growth (FIG. 5a).

[0096] <4-3> Confirmation of Osteolytic Bone Metastasis Inhibitory Effect

[0097] As shown in FIG. 5b, it was confirmed that FSTL1 of the present invention suppressed bone metastasis by about 67% or more (FIG. 5b).

<Experimental Example 5> Confirmation of Inhibitory Effect of FSTL1 on Osteoclastogenesis

[0098] <5-1> Culture of Mouse Bone Marrow Cells

[0099] ICR mouse (6-9 weeks, male) was cervical dislocated and disinfected with 70% ethanol. The skin of the tibia part was dissected and the attachment muscles were removed. The tibia was cut and the patella was dislocated to remove the tibia. The bone was cut a little bit and a 25 G needle was inserted into one end of the bone, and -MEM was poured to collect the bone marrow cells in the test tube. After centrifugation, the cells were suspended in -MEM and 2 times of Gey's solution were added to remove the red blood cells. After centrifugation, the cells were resuspended in -MEM containing 10% FBS.

[0100] <5-2> Confirmation of Inhibitory Effect on Osteoclastogenesis

[0101] The bone marrow cells cultured in the above <5-1> were inoculated in a 96-well plate at 110.sup.3 cells. Then, after FSTL1 CM of the present invention was pretreated, RANKL was treated, and after 7 days of incubation in a 5% CO.sub.2 incubator, stained with TRAP (tartrate resistance acid phosphatase), and the number of osteoclast differentiated cells was counted to confirm the inhibition of osteoclast differentiation by FSTL1 CM.

[0102] As a result, as shown in FIG. 6, it was confirmed that the FSTL1 CM of the present invention significantly inhibited osteoclast formation (FIG. 6).