KR102434325B1 - Biomarker for diagnosis or prognosis prediction of metastatic breast cancer and use thereof - Google Patents

Abstract

The present invention relates to a marker for diagnosing or predicting metastatic breast cancer and its use, and more particularly, to a marker composition for diagnosing or predicting metastatic breast cancer comprising the FBXL16 gene or a protein encoded by the gene, and metastatic breast cancer diagnosis or using the same It relates to a composition for predicting prognosis, a method for providing information for diagnosing metastatic breast cancer or predicting the prognosis of breast cancer metastasis. The present invention confirmed that FBXL16 inhibits epithelial-mesenchymal transition and angiogenesis through binding to HIF1a, thereby reducing cancer cell metastasis, stem cell ability, and angiogenesis. Accordingly, FBXL16 according to the present invention can be used as a therapeutic agent for metastatic breast cancer.

Description

Marker for diagnosis or prognosis prediction of metastatic breast cancer and use thereof

The present invention relates to a marker for diagnosing or predicting metastatic breast cancer and its use, and more particularly, to a marker composition for diagnosing or predicting metastatic breast cancer comprising the FBXL16 gene or a protein encoded by the gene, and metastatic breast cancer diagnosis or using the same It relates to a composition for predicting prognosis, a method for providing information for diagnosing metastatic breast cancer or predicting a prognosis, and the like.

Breast cancer is the second most common cancer after lung cancer, and is the fifth most common malignancy in mortality. Although there are various causes of breast cancer worldwide, the known cause is only about 20%. For the remaining 80%, the cause is unknown, but the opinion is that it is highly likely to be caused by an external environment. Diseases caused by the external environment have so many different causes that it is difficult to explain with one cause. Therefore, it is accepted that the complex external environment is the main cause of breast cancer. Diseases caused by external environmental causes cannot be predicted and prevented in advance, and the treatment process takes place only after the diagnosis of the disease is made. Early detection is more important than other cancers because it is difficult to cure when it invades the lymph nodes or metastasizes to the lymph nodes, and research on this is actively underway (Republic of Korea Patent No. 10-1966493).

On the other hand, heterogeneous breast cancer cells have different subtypes according to the expression patterns of various biomarkers, and are undergoing radiation therapy as well as drug treatment accordingly.

It is known that subtypes according to the expression pattern of breast cancer cells are divided according to the presence or absence of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2). The type that has all three hormone receptors (ER+, PR+, HER2+) is called the luminal type, and although drug therapy targeting the receptors is being carried out effectively, tissues that are negative for the three receptors (ER-) , PR-, HER2-), not only is it difficult to treat with drugs, but also has a poor prognosis because of its high penetration and metastasis compared to the luminal type. Accordingly, there is a need for a new target capable of treating metastatic breast cancer according to the above-described breast cancer cell expression pattern.

Republic of Korea Patent Registration 10-1966493 (2019.04.01)

In order to solve the above problems, the present inventors studied a target for treating breast cancer cells, and selected the E3 ligase FBXL16, which has a high expression level in luminal breast cancer, and cancer stems according to the expression of FBXL16. The present invention was completed by confirming the cell ability and the ability to inhibit cancer metastasis.

Accordingly, an object of the present invention is to provide a marker composition for diagnosing or predicting metastatic breast cancer, including the FBXL16 gene or a protein encoded by the gene.

Another object of the present invention is to provide a composition for diagnosing or prognosis of metastatic breast cancer, including an agent for measuring the level of mRNA or protein encoded by the FBXL16 gene, and a kit comprising the composition.

Another object of the present invention is to provide an information providing method for diagnosing or predicting prognosis of metastatic breast cancer, comprising measuring the expression level of the mRNA of the FBXL16 gene or the protein encoded by the gene in a biological sample derived from a subject. do it with

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating metastatic breast cancer, comprising the FBXL16 gene or a protein encoded by the gene as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a marker composition for diagnosing or predicting metastatic breast cancer, including the FBXL16 gene or a protein encoded by the gene.

In addition, the present invention provides a composition for diagnosing or predicting metastatic breast cancer, comprising an agent for measuring the level of mRNA or protein encoded by the FBXL16 gene.

In one embodiment of the present invention, the agent for measuring the mRNA level of the gene may be sense and antisense primers or probes complementary to the mRNA of the gene.

In another embodiment of the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by the gene.

In addition, the present invention provides a kit for diagnosing metastatic breast cancer or predicting a prognosis, comprising the composition.

In addition, the present invention provides an information providing method for diagnosing or predicting prognosis of metastatic breast cancer, comprising measuring the expression level of the mRNA of the FBXL16 gene or the protein encoded by the gene in a biological sample derived from a subject.

In one embodiment of the present invention, the mRNA expression level is nanoString nCounter analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real) -time PCR), RNase protection assay (RPA), microarray, and northern blotting (northern blotting) can be measured through one or more methods selected from the group consisting of.

In another embodiment of the present invention, the protein expression level is western blotting (western blotting), radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation) ), flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and at least one method selected from the group consisting of a protein chip can be measured through

In addition, the present invention provides a pharmaceutical composition for inhibiting angiogenesis or a pharmaceutical composition for preventing or treating metastatic breast cancer, comprising the FBXL16 gene or a protein encoded by the gene as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition inhibits the expression of angiopoietin1, angiopoietin2, PDFG or FGF2 or the migration and invasion of breast cancer cells. ) may be inhibiting the

In another embodiment of the present invention, the composition may induce degradation of HIF1a protein.

In addition, the present invention provides a method for screening a therapeutic agent for metastatic breast cancer.

The present invention confirmed that FBXL16 inhibits epithelial mesenchymal transition and angiogenesis through binding to HIF1a, thereby reducing cancer cell metastasis, stem cell ability, and angiogenesis. Accordingly, FBXL16 according to the present invention can be used as a therapeutic agent for metastatic breast cancer.

Figure 1 is the result of confirming the expression screening of the E3 junction gene in breast cancer cells, Figures 1a and 1b confirm the expression of the E3 junction gene through the METABRIC and GSE database, Figure 1c is in each subtype of breast cancer through the TCGA database It is a result of comparing the expression level of FBXL16, Figure 1d is the confirmation of the expression of FBXL16 according to the breast cancer type through the GSEA patient database, Figure 1e is the expression of FBXL16 according to the breast cancer type through the UCSC HUB and GEO data cell line analysis. 1f shows the measurement of the expression level of FBXL16 through Western blot and RT-PCR, and FIG. 1g shows the results of Kaplan-Meier survival analysis for breast cancer patients.
Figure 2 relates to the inhibition of invasiveness and angiogenesis of breast cancer by FBXL16. Figure 2a shows the degree of epithelial-mesenchymal transition according to the expression of FBXL16 through the GSEA database, and Figure 2b shows the overexpression of FBXL16 in luminal type cells. After confirming the change in epithelial mesenchymal transition, FIG. 2c is confirming the angiogenesis change according to FBXL16 expression through the GSEA database, and FIG. 2d is the FBXL16 expression after co-culturing HUVEC cells and breast cancer cells through tube formation assay. Shows the results of checking the degree of angiogenesis.
Fig. 3 shows the interaction between FBXL16 and HIF1a. Figs. 3a and 3b are the results of confirming the binding relationship between FBXL16 and HIF1a through immunoprecipitation, and Fig. 3c is the result of immunoprecipitation after expression of FBXL16 and HIF1a. Shows the results, Figure 3d shows the binding of FBXL16 and HIF1a confirmed through PLA staining, Figure 3e shows the results of observing the half life of FBXL16 and HIF1a through CHX assay.
Figure 4 confirms that FBXL16 is involved in epithelial mesenchymal transition and angiogenesis by regulating HIF1a in breast cancer. 4b shows the epithelial mesenchymal transition and the degree of angiogenesis according to FBXL16 alone expression and FBXL16 and HIF1a expression through aorta assay, and FIG. 4c shows the epithelial mesenchymal transition according to FBXL16 single expression and FBXL16 and HIF1a expression through ICC. and the results of confirming the degree of angiogenesis.

As a result of studying a target for treating breast cancer cells, the present inventors selected the E3 ligase FBXL16, which has a high expression level in luminal breast cancer, and confirmed the cancer stem cell ability and cancer metastasis ability according to the expression of FBXL16. The present invention was completed.

Accordingly, the present invention provides a marker composition for diagnosing or predicting metastatic breast cancer, including the FBXL16 gene or a protein encoded by the gene.

In addition, the present invention provides a composition for diagnosing or predicting metastatic breast cancer, comprising an agent for measuring the level of mRNA or protein encoded by the FBXL16 gene, and a kit for diagnosing or predicting metastatic breast cancer comprising the same.

In the present invention, the FBXL16 gene may consist of the nucleotide sequence of SEQ ID NO: 2, and homologues of the nucleotide sequence are also included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence of SEQ ID NO: 2, respectively. can

In addition, the protein encoded by the FBXL16 gene may consist of the amino acid sequence of SEQ ID NO: 1, and is interpreted to include a sequence showing substantial identity to the sequence shown in SEQ ID NO: 1. The substantial identity is at least 80% when the sequence of the present invention and any other sequence are aligned to the maximum correspondence, and the aligned sequence is analyzed using an algorithm commonly used in the art. A sequence exhibiting homology, more preferably 90%, 91%, 92%, 93%, 94%, 95% homology, most preferably 96%, 97%, 98%, 99% homology. it means.

"Metastatic breast cancer", which is a disease targeted by the present invention, is one of the types of breast cancer, and compared to the luminal type, it has high penetrability and is negative for three hormone receptors that metastasize easily (ER-, PR-, HER2-). ), in which transformed, i.e., cancerous cells derived from at least one primary breast tumor are separated from the primary tumor and separated from the primary tumor (hereinafter referred to as "remote site") As a disease that continues to grow into cancer in , the remote site may be, for example, in the same breast (ipsilateral breast) or different breast (contralateral breast) where the primary tumor is located. Also, for example, the remote site may be within one or more lymph nodes, which may be mobile or stationary, may be ipsilateral or contralateral to the primary tumor, and may be above the clavicle, in the armpit, or the like.

As used herein, the term “prediction of prognosis” means that it can predict the risk of breast cancer metastasis as well as the function of predicting cell viability and cell proliferation caused by breast cancer.

As used herein, the term "diagnosis" in a broad sense means judging the actual condition of a patient's disease in all aspects. The content of the judgment is the disease name, etiology, disease type, severity, detailed mode of the disease, and the presence or absence of complications. Diagnosis in the present invention is to determine whether or not metastatic breast cancer is onset and the level of progression.

In the present invention, the agent for measuring the mRNA level may be sense and antisense primers or probes that complementarily bind to the mRNA of a gene.

As used herein, the term “primer” refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, etc. as a short gene sequence serving as a starting point of DNA synthesis. The primers can be synthesized and used with a length of typically 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, etc. by a known method.

As used herein, the term "probe" refers to a nucleic acid capable of specifically binding to mRNA of several bases to several hundred bases in length produced through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be checked by labeling radioactive isotopes or enzymes, and can be designed and modified by a known method.

In the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene, but is not limited thereto.

As used herein, the term "antibody" includes immunoglobulin molecules having immunological reactivity with a specific antigen, and includes both monoclonal and polyclonal antibodies. The antibody also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

The kit for prediction of the present invention consists of one or more other component compositions, solutions or devices suitable for the analysis method.

For example, the kit of the present invention comprises genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, an appropriate amount of a DNA polymerase, a dNTP mixture, a PCR buffer, and water to perform PCR. It may be a kit comprising The PCR buffer may contain KCl, Tris-HCl and MgCl ₂ . In addition, components necessary for performing electrophoresis that can confirm whether or not the PCR product is amplified may be additionally included in the kit of the present invention.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included. In addition, a primer pair specific for a gene used as a quantitative control may be included.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative structural gene or fragment thereof. In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.

In addition, the kit of the present invention may be a kit characterized in that it includes essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for a marker protein, and an agent for measuring the protein level. The ELISA kit may include a reagent capable of detecting an antibody that has formed an "antigen-antibody complex", for example, a labeled secondary antibody, chromopores, an enzyme, and a substrate thereof. In addition, an antibody specific for the quantitative control protein may be included.

As used herein, the term “antigen-antibody complex” refers to a combination of a protein encoded by a gene and an antibody specific thereto. The amount of formation of the antigen-antibody complex can be quantitatively measured through the magnitude of the signal of the detection label. The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto.

In another aspect of the present invention, the present invention provides an information providing method for diagnosing or predicting prognosis of metastatic breast cancer, comprising measuring the expression level of the mRNA of the FBXL16 gene or the protein encoded by the gene in a biological sample derived from a subject do.

The term "information providing method for diagnosis or prognosis of metastatic breast cancer" used in the present invention is a preliminary step for diagnosis and provides objective basic information necessary for diagnosing metastatic breast cancer or predicting the risk of metastasis of breast cancer. of clinical judgment or findings are excluded.

The biological sample derived from the subject may include, but is not limited to, tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.

The expression level of the mRNA is NanoString nCounter analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection It may be measured by at least one method selected from the group consisting of RNase protection assay (RPA), microarray, and northern blotting, but is not limited thereto.

The protein expression level is determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation according to a conventional method known in the art. ), flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and at least one method selected from the group consisting of a protein chip It may be measured through, but is not limited thereto.

The present inventors have identified the use of FBXL16 according to the present invention as a novel target for diagnosing and treating metastatic breast cancer through specific examples.

In one embodiment of the present invention, as a result of confirming the expression level of FBXL16 for each breast cancer type, the expression of FBXL16 was significantly high in luminal tissues with relatively few metastases, and patients with high FBXL16 expression for breast cancer patients had a better breast cancer prognosis. It was confirmed that it appeared (see Example 1).

In another embodiment of the present invention, as a result of confirming the effect of FBXL16 on metastasis and angiogenesis of breast cancer cells, it was confirmed that epithelial-mesenchymal transition and angiogenesis were reduced in FBXL16-overexpressed cells (see Example 2).

Further, in another embodiment of the present invention, in order to confirm that FBXL16 is involved in epithelial mesenchymal transition and angiogenesis by regulating HIF1a, epithelial mesenchymal transition and blood vessels inhibited by FBXL16 through invasion/migration, aorta assay, and ICC. By confirming that angiogenesis was increased when HIF1a was re-expressed, it was confirmed that FBXL16 could act as a tumor suppressor by inhibiting HIF1a expression (see Example 4).

Accordingly, as another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating metastatic breast cancer, comprising the FBXL16 gene or a protein encoded by the gene as an active ingredient.

As used herein, the term “prevention” refers to any action of inhibiting or delaying the onset of metastatic breast cancer by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of metastatic breast cancer are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention includes the FBXL16 gene or a protein encoded by the gene as an active ingredient, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in the formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, but preferably may be administered orally, and the dosage Although it varies depending on the condition and weight of the patient, the degree of disease, the drug form, the administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical 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 or diagnose a disease at a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by the patient's disease type, severity, and drug activity, sensitivity to drugs, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination, generally 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight, may be administered daily or every other day, or may be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

In addition, according to the present invention, it was confirmed that FBXL16 can inhibit angiogenesis by inducing the degradation of HIF1a or inhibit the penetration and migration ability of breast cancer. Among unknown substances, substances that increase the binding of FBXL16 and HIF1a By selecting , a therapeutic agent for metastatic breast cancer can be selected.

Accordingly, as another aspect of the present invention, there is provided a method for screening a therapeutic agent for metastatic breast cancer, comprising the following steps.

(a) treating cells with a candidate substance in vitro;

(b) measuring the binding level of FBXL16 and HIF1a in the cells; and

(c) selecting a substance that increases the binding level of FBXL16 and HIF1a compared to the candidate substance untreated group.

The cell may be a breast tissue-derived cell, but is not limited thereto.

The candidate material may be selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids, and peptides, but is not limited thereto.

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

[Example]

Example 1. Screening for expression of E3 ligase in breast cancer cells

As a result of analysis through the METABRIC and GSE databases, as shown in FIGS. 1A and 1B , it was confirmed that the FBXL16 gene showed the highest expression level in the luminal type among various E3 zygote genes.

In addition, as a result of comparing the expression level of FBXL16 in each subtype of breast cancer cells through the TCGA database, it was confirmed that the expression level of FBXL16 was highest in the luminal type as shown in FIG. 1c, and using the GSEA patient database As a result of the analysis, as shown in FIG. 1d , it was confirmed that the expression of FBXL16 was higher in normal breast cancer than in triple-negative breast cancer, and in UCSC HUB and GEO data cell line analysis, FBXL16 was the highest in ER+ type. As a result of measuring the expression of FBXL16 through Western blot and RT-PCR, it was confirmed that the expression level was expressed, and as shown in FIG. It was confirmed that the expression of FBXL16 was high in the strong breast cancer cells.

Furthermore, as a result of confirming the correlation between FBXL16 expression and the survival rate of breast cancer patients through Kaplan-Meier survival rate analysis, as shown in FIG. 1E , the expression of FBXL16 is inversely proportional to the survival rate of the patient. Since the prognosis of FBXL16 is good, it can be inferred that FBXL16 can act as a tumor suppressor that inhibits the malignancy of cancer.

Example 2. Confirmation of breast cancer invasion capacity and angiogenesis inhibition according to FBXL16 expression

2-1. Confirmation of breast cancer invasive ability according to FBXL16 expression

In order to determine whether FBXL16 affects breast cancer cell invasion and epithelial mesenchymal transition, the degree of epithelial mesenchymal transition according to the expression of FBXL16 was analyzed through the GSEA database. It was confirmed that the liver lobe transition was lowered.

In addition, as a result of analyzing the degree of epithelial-mesenchymal transition after overexpressing FBXL16 in luminal type MDA-MB-231 cells through invasion migration assay and western blot, as shown in FIG. 2b, when FBXL16 was overexpressed compared to the control group, invasion And it was confirmed that the migration is reduced, and using Western blot and immunocytochemistry methods, E-cadherin, Vimentin, Fibronectin, which are major markers of epithelial-mesenchymal transition, and regulation As a result of checking the expression levels of Zeb1, Snail, and Slug, which are known factors, it was confirmed that the expression of major markers and regulators of epithelial-mesenchymal transition was decreased when FBXL16 was expressed.

2-2. Confirmation of angiogenesis inhibition by FBXL16 expression

In order to confirm the effect of FBXL16 on angiogenesis of breast cancer cells, the degree of angiogenesis according to the expression of FBXL16 was checked through the GSEA database.

In addition, as a result of co-culture of HUVEC cells and breast cancer cells MDA_MB_231 through tube formation assay to confirm the degree of angiogenesis according to FBXL16 expression, as shown in FIG. 2d, angiogenesis was observed in FBXL16-expressing MDA_MB_231 cells. It was confirmed that it was significantly reduced.

Furthermore, as a result of confirming the change in the expression of growth factors involved in angiogenesis according to the expression of FBXL16, it was confirmed that when FBXL16 was expressed, the expression of ANG1, ANG2, PDGF, and FGF2 was decreased as shown in FIG. 2e.

From the results of Example 2, it can be inferred that FBXL16 can inhibit invasiveness and angiogenesis in breast cancer cells.

Example 3. Confirmation of interaction between FBXL16 and HIF1a

After regulating the gene expression of FBXL16, a tentative target of FBXL16 was set through immunoprecipitation assay, and HIF1a was selected as a direct target through gst-pull down assay.

based on the selected target. In order to investigate the binding action of FBXL16 and its substrate HIF1a in breast cancer cells, MDA_MB_231 and MCF7 cells of breast cancer cells were analyzed by immunoprecipitation. .

In addition, when FBXL16 and HIF1a were expressed in HEK293T cells through immunoprecipitation, it was confirmed that FBXL16 and HIF1a, a substrate, were bound to each other as shown in FIG. 3c.

Furthermore, as a result of analyzing FBXL16 and HIF1a through PLA staining, it was confirmed that FBXL16 exhibited direct binding to HIF1a as shown in FIG. 3d. As a result of observing half life through CHX analysis, as shown in FIG. 3e, It was confirmed that HIF1a was degraded faster than the control by FBXL16.

From the above results, it can be inferred that FBXL16 degrades HIF1a in breast cancer.

Example 4. Confirmation of inhibition of epithelial-mesenchymal transition and angiogenesis by the interaction of FBXL16 and HIF1a in breast cancer

HIF1a is degraded by FBXL16, and in order to check whether malignancy of breast cancer cells is suppressed based on the above degradation action, FBXL16 was expressed in luminal breast cancer cells, then HIF1a was expressed again, and invasion migration assay, aorta assay and ICC were performed. confirmed through.

As a result, as shown in FIGS. 4A to 4C , it was confirmed that FBXL16 inhibited epithelial mesenchymal transition and angiogenesis, but increased epithelial mesenchymal transition and angiogenesis again when HIF1a was expressed.

From the above results, it was confirmed that FBXL16 could act as a tumor suppressor by inhibiting the expression of HIF1a in breast cancer cells.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Biomarker for diagnosis or prognosis prediction of metastatic breast cancer and use thereof <130> PD20-105 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 479 <212> PRT <213> FBXL16 <400> 1 Met Ser Ser Pro Gly Ile Asp Gly Asp Pro Lys Pro Pro Cys Leu Pro 1 5 10 15 Arg Asn Gly Leu Val Lys Leu Pro Gly Gln Pro Asn Gly Leu Gly Ala 20 25 30 Ala Ser Ile Thr Lys Gly Thr Pro Ala Thr Lys Asn Arg Pro Cys Gln 35 40 45 Pro Pro Pro Pro Pro Thr Leu Pro Pro Pro Ser Leu Ala Ala Pro Leu 50 55 60 Ser Arg Ala Ala Leu Ala Gly Gly Pro Cys Thr Pro Ala Gly Gly Pro 65 70 75 80 Ala Ser Ala Leu Ala Pro Gly His Pro Ala Glu Arg Pro Pro Leu Ala 85 90 95 Thr Asp Glu Lys Ile Leu Asn Gly Leu Phe Trp Tyr Phe Ser Ala Cys 100 105 110 Glu Lys Cys Val Leu Ala Gln Val Cys Lys Ala Trp Arg Arg Val Leu 115 120 125 Tyr Gln Pro Lys Phe Trp Ala Gly Leu Thr Pro Val Leu His Al a Lys 130 135 140 Glu Leu Tyr Asn Val Leu Pro Gly Gly Glu Lys Glu Phe Val Asn Leu 145 150 155 160 Gln Gly Phe Ala Ala Arg Gly Phe Glu Gly Phe Cys Leu Val Gly Val 165 170 175 Ser Asp Leu Asp Ile Cys Glu Phe Ile Asp Asn Tyr Ala Leu Ser Lys 180 185 190 Lys Gly Val Lys Ala Met Ser Leu Lys Arg Ser Thr Ile Thr Asp Ala 195 200 205 Gly Leu Glu Val Met Leu Glu Gln Met Gln Gly Val Val Arg Leu Glu 210 215 220 Leu Ser Gly Cys Asn Asp Phe Thr Glu Ala Gly Leu Trp Ser Ser Leu 225 230 235 240 Ser Ala Arg Ile Thr Ser Leu Ser Val Ser Asp Cys Ile Asn Val Ala 245 250 255 Asp Asp Ala Ile Ala Ala Ile Ser Gln Leu Leu Pro Asn Leu Ala Glu 260 265 270 Leu Ser Leu Gln Ala Tyr His Val Thr Asp Thr Al a Leu Ala Tyr Phe 275 280 285 Thr Ala Arg Gln Gly His Ser Thr His Thr Leu Arg Leu Leu Ser Cys 290 295 300 Trp Glu Ile Thr Asn His Gly Val Val Asn Val Val His Ser Leu Pro 305 310 315 320 Asn Leu Thr Ala Leu Ser Leu Ser Gly Cys Ser Lys Val Thr Asp Asp 325 330 335 Gly Val Glu Leu Val Ala Glu Asn Leu Arg Lys Leu Arg Ser Leu Asp 340 345 350 Leu Ser Trp Cys Pro Arg Ile Thr Asp Met Ala Leu Glu Tyr Val Ala 355 360 365 Cys Asp Leu His Arg Leu Glu Glu Leu Val Leu Asp Arg Cys Val Arg 370 375 380 Ile Thr Asp Thr Gly Leu Ser Tyr Leu Ser Thr Met Ser Ser Leu Arg 385 390 395 400 Ser Leu Tyr Leu Arg Trp Cys Cys Gln Val Gln Asp Phe Gly Leu Lys 405 410 415 His Leu Leu Ala Leu Gly Ser Leu Ar g Leu Leu Ser Leu Ala Gly Cys 420 425 430 Pro Leu Leu Thr Thr Thr Gly Leu Ser Gly Leu Val Gln Leu Gln Glu 435 440 445 Leu Glu Glu Leu Glu Leu Thr Asn Cys Pro Gly Ala Thr Pro Glu Leu 450 455 460 Phe Lys Tyr Phe Ser Gln His Leu Pro Arg Cys Leu Val Ile Glu 465 470 475 <210> 2 <211> 15354 <212> DNA <213> FBXL16 <400> 2 caaagttgcc cggcacaggt ggggtcacag aggtggaggg taggtgggtc ggtggaggg taggtgggtc ggagagggtggtgggtc acagtggtagagtg 60 gg 120 agctggtgac agaggtggag ggtaggtggg tcacagctgg tgacagaggt ggagggtagg 180 tgggtcacag ctggtgacag aggtggaggg taggtgggtc acagctggtg acagaggtgg 240 ggggtaggtg ggtcacagct ggtcacagag gtggagggca ggtgggtcac cgctttggag 300 gagcacggtt ggcctttctg tgatgatctt ccacacactc ttccctgctc agattcccca 360 caggcagggc tagcttcttg ggcctcgcca gggttgcatg acccaaatgc ctgactccca 420 cgctcaccct atgcacttgg ggtgtgtcca gcccacccta tgcacttggg gtgtgtccag 480 cctgccctgt gcgcttgggg tgtgtccggc tccagcccca gctgggactc aacacaagca 540 cagcttgttc tctgcctgag gccctggcac ctgctgtggt gagagcccca ggcacgaggt 600 gaaaccctcc taccgggagc agtccacgcc tggggtggaa gctgctgcca gagggccagc 660 ctgccagacc atgcaccgga gacacccaga taggttggac cgccgctccc ctgttgcccc 720 aactgtcttc acggggctcg tcctgtctgc cccactccgg gcaccttctt tggtcagtat 780 ccaggtaccg ggcaagactt acgcagaaga gctgctgcgc tcaccccagg tagacccagt 840 ggcgccccca cagcaagcac agaaccaccc caccccggga cccagccagg gtgccgtcag 900 tctgaccctc cttcccgctg accccaggcc tcacaccggc cccttcggcc aggcttccca 960 tcaggacgct gggaacagca gctggtttca ctcctattga gatactctca ggagccccat 1020 tcagccctgt agctctttcc cggtgcaaag aacccctctg ctctccccac acctgctccc 1080 tcccccggcc ctgctcccag gagaccccag gatagggcgc cattggcctc cattctcctg 1140 ccctgagcct gctggggcgt ctggggtctc cagcggtccc caccgagtcc tccacctaac 1200 ctagcacgcc ccacccctgc cccgggctgc agcagccgcc gccgcctcca gcctccagtt 1260 tattcccgag ggaacttgcc aggctcctcg gatctttgca cccccattcc cgagccatcc 1320 ctccctcgcg ccgtgggggg gtctcagctc cagtggggca ccctcaggaa gcccccgcca 1380 gaagtcacca ccgcaagccc tcgggtgctt tcactgtcac tgtctgaact gatccgtgca 1440 gtcacttgtt ttcgcgctct gcgggtgtgc cagggtctcc aatgaggggc ttccggccgg 1500 gcacgcagca ggtgcgcggg gggtgtgagg gctgtgaagg gtccttgccg tgggctgggc 1560 gggagggtcc agagacgggg gtctgaccac cctgtctcct ctgggccggg tctcgccttc 1620 ctcgcctccc caagtggtgg ccaagggccg ggacgcgcgg tgccgccccg gtccccatag 1680 gaccccggac ccctgggcca cccgccggcg cctgctctcc cgcctcccct cgccctccct 1740 tctgcctccc gcgccctccc cgcccgccgc gcgccggggt ttgttattgt gcgggtgccc 1800 gcggcgagcg gggcgggggc gcgtgccggg cctggcgcgt gctcgtggac gccgtgccgg 1860 gagcgcgccg cgcgcggggg ccggggcggg gcgggggcgg ggctcggcat tctcggcgcc 1920 cccgcccggg ctcgcgaacc cggattggct cgccacgccg ggagcgcgcg agggcgcggc 1980 gtttggtgcc gggcgggggg cgcgcgcggc gccggcggcg gccacggagg agcgcgggga 2040 gggcgagggg aggaggaggc gcccgcctgg ttccctgcaa agcggcctta tttatctggg 2100 cacagcctca gcctccccgg tgggaggctt ggggcggccg atcctctccc accggggagc 2160 tcctttccgt gcgct gccga gggggcccgg ccaggacggg acgcggggcg cagggcgcgg 2220 ccgggccctg ccggccagtc cagcaccccg gcggcccccg cgcgccgacg cccatgccgg 2280 gctccccatg acgggaggtc gcccggcgga cagcgtggca tgagccagga gcccgggccg 2340 aggtaaggtg cgcgtgcggc cgggctgcgg ggccgggagg gggccaggcc gcacctccgc 2400 ggcggccaca atgaggaggg ggcggggtgg gggtgggggc cgcgccgccg ccgcccggga 2460 agggcttacg taacggcagc cccttcccca cctcccccgc ggccgctggg cctcggtccc 2520 tgcctggcct tcccgcagcc ccgcgccccg gcccctggcc cggaagaggg ccgctcccca 2580 ctggccactc ggccggggct gggggtccgt gcctcctggg acgccgaaag gaggaattgt 2640 aggcccccgg cctccacccc gggcgcctgg tcggctcaga gggcgccccc ggaccgaagt 2700 gacaggccag agaccgttaa actgcatggg ggtcgcgtgg atgtgcggcc tgaggggcgg 2760 ccctccccgg gcgggcggct tctggacccg gggcctgcgc tgagctactg cccgcgggag 2820 gaggagggtg gggggaggct ggcaccttcc tggttctgga ggcgggaccg ggccttggcc 2880 aaggttggag ggcctcagaa gggaaacggc accttcagac ctgctcggga ggggtccggt 2940 gtcctcctgg ggattaggtg ggtggagagc gttccccacc catcacctca ggagagaggg 3000 ctgcggtggc agcttcagga actgacggcc cagcggccac tccaggccct tcccaggctc 3060 caggctctgt tcgggcagcc tgccctccgc aggaggagca gatggcggag gccacctcac 3120 ctgcctcttc cttcaggccc ttcccccaca cccgtgcatc cgagggccca atggccaggg 3180 gcagctcagg cctccctgga cacagggccg tctcccagcc caccccctct gtggggtgct 3240 ggacagcagc cttggagcag ataggcctgg gtcaggccag gagactgaag ggttttctgt 3300 ctcccggacc agggcagagg cctagtggaa cctggctggt ctggccagag gtggacacag 3360 ggctggacct ggagccgcct ggaggggagg gaaattcaag gcccagctgc aaacatcgct 3420 gtgtggggga accctaggac tcacttgccc cactccctgc cccctgccac cccaactgcc 3480 cctcatctgg ggccagaccc ttgagttcag cccctccatc gacagactca ctgcctccag 3540 ctcacaccca cttccggggc ggctggacga gtcctacctc ccactgatga gaaggacaca 3600 aacagcttcc tgtgtgggag gcacggggcc cttgaaccag ctggacatgg acccaggaag 3660 ccttgtgttg ctgagagtta gggatctccc acccaggtgg cactgttcag ccacccgaag 3720 accccatctc cttgcatgtg ggcacccact gctgtgcctc ccatgactgg attggcagtg 3780 aagggagctc tgtgggtgag caggtgactg agccttcccc accagtggtg caggaggagt 3840 caacggccaa actggctgca atgtgt gggt tcaggcctca ttccttcact gggcaggaaa 3900 gaggggtgat ggttagtggc agctgcctgg agcagtaggc actaggggcc caggcctcgc 3960 ctctgagggt gctcatgctc ttccgagcag gggctgagat gaggcctgga tcccagcacc 4020 cactccccag ggtgtccagc tttcaaggct gagctgccct gggcttgacc atgggccctc 4080 agtctgctgg ggcagcaaca cgggtttctc ttgaggccac gtgcctgctg cgccatggtg 4140 cccctcctca gagcccctgg tgcaccccca gaagcatctc aaactggggg cgggagagga 4200 gctctgccgc tgtgccacgt gccaagtggc agggacgcgg gtgtgcctgc gtcacggggc 4260 tgccacggaa tgtgtcagtc tgcaccctgt ccccaatccc cagccacccc agctgtcagc 4320 caggctctgg ggctgccact gcagatggag cccgatgtgc atttctctgg ccaccagggc 4380 cctgcccagc tctaaggccc tctgcaggca gggctcaatg gtccctgagg ttctatgagg 4440 gctgagtggt tggttaggct gtgccccagt ctggtgctga cagagatggg ggcaccagag 4500 atgggggaag agaccttcaa actgggggcg ctgaggggtg aggaggtttc ccaggcatgt 4560 gtccaaaagg atgtcccagg ggtgggcacg cggtgtgcag cctctgcaag gaatgcagcg 4620 ccgtggcagg gaccctgcag gatgagggca gtacttggag agaccactag cccagggccc 4680 agcaggaagg catcgagacc ccatgaggca g aacagggca gagcgaggac cctgtgaggt 4740 acacctggca ggacccaggt agagggaatc agccagcccc ggggcttttg gagccaatcc 4800 tagctctatg taacttctgg aaaatccgtc tgcaagctct gcttccccac tcctagttag 4860 gcagtgtgtg aaggaaggac tctggccggt cagaggaggg gtgcgtgtgg tgccagggag 4920 agcggggcag gaggaccccc ttccctctct tggctcaggc cagatggtgg aggacgggca 4980 gccacttcct cagccctgca gctcctgaag agagagccag ctgaaaagga gatgggccct 5040 cctcatccga aggctctgtg tattgcaagg cccaccccag ccctggggtc ttgggagccc 5100 tctcagtgac tcaggtacca acctagtccc aaccccagag gccactctcc ccgcggacac 5160 tggccatggc acgatccggg ttaatttccc gagctgtgcc ttccccgctg accggcctct 5220 tcctaaattt gaccaatcct ccatcagccc tccgggggcc ttggcagctg cacccctttc 5280 tgcgtgactg agctagcgga gccacattcc gcattcattc attcgttccc taattcattc 5340 attccgcgaa catgcagagg ccatgcacgt tggtgctgtc tgtcctcaag agtcctcagg 5400 ccaggaggag agacaccagg tggacagtga tgataaccca tgtgattgca agaccctgga 5460 gacgctttct caagagctgg gggtcccgga gcaggaggca ggggcgacct cgcaccagga 5520 actgagggat ggaggagaag agcttgcagg gctaagg cag ggaaaccctg ccaggcaagc 5580 agggggaacg gcgtgtgccc aggcagagga gcaggggagg aggaatggca tggctgggct 5640 gtggcatcag ggctgggaag gggccggagc tgaggtctcc gggctcagtg acttgattcg 5700 cagaagaggc agctcctgcg gggcccccac atggccgtct gggaatggtc agcctggatg 5760 ctgtacatgt acatgtgggg acccccagga gcacccaggc cccaccgtgc tccctaggct 5820 cactctggcc acacgcttca cactgcgtcc cctggggagg acacagaggc cacggccacc 5880 tctccccagc agctcccagg ccaggctcag gctgctgagg acaagacagc tgaggtgggg 5940 ccacctctct ctctttcccc tggggtgagc cccggagcct tgacaggatt ccacttcaag 6000 ctgagccagc accacagggg ctgaggctga ggatggtgca ggcatggcca cctttttaac 6060 aagtctcttc cccgtattag cacccgggct gcttgtccct aaagggagag gcagtcccta 6120 gcagcccacg caggtcgtcc gagcccctct gggtggaacg agggctctca cagtcgccaa 6180 gcaggaaagg gggctctgtg agccatggga accggctcct tggtgaccgt ctggctgggt 6240 gggtggggcc gggcccaggg ccaagcagag gggcccttgt gtgagccccc agggacttct 6300 cgctgaacgg gcagagagac cccagtctcc agttgagctc agctttctcc cgcagcgccc 6360 atgcctgggc caaagtgtgt ggctgtttgc atttttgagc ta cggggtgg gggatattcg 6420 ggctgagtca gtcaagccgg gagtgtgagg agcagcgggg gccggcagga acgtggacgc 6480 gagattgggg agcagtgggg ctgcctgtgc ggggatgcag ctcccaccaa gcacacgggt 6540 cgagggctga ccacaggcag agcagcgcct tctggcagcg ctgcgggtgt ggatgaggga 6600 agccaggccc cggacccata cccagacggc cgctcctcag cctgtcccct ccctcctgtg 6660 ggctctcaaa gtgcggcttg gtttctccat ctcgaccctg aggcctggag gtggggacgg 6720 gactgtgggc gccggcaaac gtgagctccc gagcggggcg ggaaaggagc cccgcgaatg 6780 cctcccccag ccccggcgct ccacgtgcag gcccatgact gggagcgttt tcaacctaaa 6840 tcggcactgc gccgggatta ttcctcccct ggcgccgccc ccgccgcctc ctccggatcc 6900 ttcccggctt ctcccccact cttcgccccc ggggcccctc cctgctgagc gggggctgcc 6960 gggatttggg attgcggggc tgccccgacc ccgcagccgt ccgcggtgga agccagcccg 7020 ggcctggccc tgagcgcgcg gctggggcgg ccgctgtcca tggtgctggc gcgggtggcg 7080 cgccggccgg cggggtctgc ggaggcccag gcgtccctgg cggggaaggc ggcgcttccg 7140 ggtctgggga gcgagggtcc ccgctctagg ggcacggagg atatttagac tggggcgccc 7200 gtgggcctgg tgctgccgcc tcgggcgccg gcatcctgcg atatcccc tc tccccggctc 7260 tgggagggaa ccgaggcagc cccgcgccag gcctccccgg acacctgggg caaattcgct 7320 cctcagagcc tcgtgttcct tccccaaacc tgaggtagcg acccctccac ccctaagggc 7380 ggccgtgagg tcgaatcatg tgccgacccc agccggccct tgcaaacgtt gtgatgtcgg 7440 cctccggagg ccgcgccttc tgatcatggg cacatgatgg gggtcaccgg ctgtgggtgc 7500 ctcctctgtg gagggcccgg cctcaccttc caggacccga atgatactct ggggaggggg 7560 agatggtaca gagggtggag ctggggctcc tgccattagg gcaggaagga ctttaaaccc 7620 aaagctgggt tctcagccac gagggccacg tgttctggcg agacccctgg tcccggctcc 7680 ggtctacacc ccctccccag atgtggaccg gtggcaaggc tggccctgtc tggggcttgg 7740 aagccagtgt ggggacctgc tgctgggaag agcggggcag gacctcccac tgcctccctg 7800 cctccctgcc tcccctcccc cctgaccaga ttatggggaa tgggctgcgg gccttggggt 7860 tgcagtgccg tccgagaccc cgccactctc tgtggtggag caggccaggc cctgtagccc 7920 cagaagggac actcagccaa aaagatgtga gccatcctct tcctgagagt ctgcatattg 7980 cagggcccac cccaagcgtt ggaatcttcg tgggagactc tcagacaccc ccttggtggc 8040 tccaagagac cttccagtga ggctgtggca ggagggggca gggtgggctg gga gaggctt 8100 tggcccctcc tctgagattt tggggcagga aggcaggacc tgcatgaggc tccaggctta 8160 gtcctggagt cacctaagac cggggaagat gcatcttgct ttgccctgga gtgtggccgt 8220 ggggctcaga catcactctc ccagaccttg ctatgagcca cgcactgagc tcgttcactt 8280 tgcaatgccc caagggagca ggtaccgtca cctccccagg cttcaaggtg aggaaattga 8340 ggctcagaga agggaagtgg cttgccaaga ccacagagcc agggtggggc ccatttcaag 8400 tccagggcgt gtgaccccag gggctctacc tcctccccgg atgtcggggt gggggcggca 8460 ggagatccga actgtggagc agatggtggc acagtgaggg tcccacctct gcttctttcc 8520 gtggctgtgc ccagtgcccc ctgccaccca cttacagact ggaggcctga ggagggtgcc 8580 cgggactgct cccactccac cctgtgcgtg gatgtcgctg gcctgaagat aacctccgca 8640 tggccttcct cagccagctc tgcccgaact ttggcttcac cacttatctg gccatgacct 8700 cgggcaggca gtttgcccag ccttggcctc agtttcctca tctgtgcagt gggtacagct 8760 atcacagcat cgttctcatt gggcagaaga ttaaagcgac agcttgtgtc cagggctaac 8820 catgctggca ccacagcaca ccctgggaag atgcccgccc tttctttgca tggattcttt 8880 tttctttttc ttttcttttt ttctttttct ttctttcttt cttttttttt ttttttttt t 8940 gagacaaagt ctcactctgt tccccaggct ggagtacagt ggtgtgatct cggctcgctg 9000 caacctccgc ctcctggatt caagtgattc tcctgcctca gcctcccaag tagctgggat 9060 tacaggagcc tgccaccaag cccggctaat ttttgtattt ttagtagaga aggggtttca 9120 ccatgttggc caggtggtct caaacccctg accttgtaat ccacccacct cagcctccca 9180 aagtgctaga attacaggca tcagccactg cacccagccc cttgggtaga ttctgagcca 9240 gggaagctcc tgtgcatgtg gagggtggct gcagagtcag caccagagcc cctcctgagc 9300 gctgtgcagt caccctgcag gggaggggcc agaggccacc ccagaccctt cccttcagtt 9360 cccctggcct gcccagaggg gtgggcctgg tgggtaccta cagtggacgg cacagcagag 9420 aggtcaagag agcagtcctg gcctcaccag gtccacctcc attttcttct ctgtaaaatg 9480 gccatcatgg tacctaagtg ctaaaaagtg tcggcgtttc ttcgcctcac tgggcaaaca 9540 aaaaggtttc cggtcaggca cggtggctca ttcccataat cccaggactt tgggaggctg 9600 aggcgggtgg atcacttgag gtcaggagtt tgagaccagc ctgaccaaca tggagaaacc 9660 ccatctctac taaaaataca aaattagccg ggtgtggtgg tgggcgcccg aattcccagc 9720 tacacgggag gctgaggcag gagaatcact tgaacctacg aggcagagag gttgcgggga 9780 gccaagatcc tgtctctgca ctccagtctg cgctccaggc aacaagagtg aaactgtttc 9840 tttttttttt ttttgagaca gagtcttgct cagttgccca ggctggagtg cagtggcgca 9900 atctcggctc actgcaagct ccgcctcccg ggttcacgtc attctcctgc tgcagcctcc 9960 caagtagctg gtactacagg cgcctgccac catgcccagc taattttttg tatttttaga 10020 agagatggtg tttcactgtg ttagccagga tggtctcgat ctcctgacct catgatccac 10080 ccgcctcggc ctcccaaagt gctgggatta caggcatgag ccaccgcgcc cggccggaac 10140 tctgtttcaa aaagaaaaaa aacaaaagaa aaagagggtg tccatgggca atgaaggttg 10200 ggctcagtgc ataccgtagg tgcccagtga gtgctgccag tggccatggt tggcttcctg 10260 ttgctgctca cagtctggga gggagaagca ggcactccca tcctctctgt ctggtggttc 10320 tgggagcacc atagggacgc ccaaggaggg aaggagcccc actgcacccg cccccaaccc 10380 cggccttcca gactcagcaa cagactcact ccccctcccg gccctcatcc acagagcgtg 10440 ccaggaagat gtcgagcccg ggcatcgacg gcgaccccaa gcctccatgc ttgcctcgaa 10500 acggtctggt gaagctgccg ggccagccca acggcctggg tgcggccagc atcaccaagg 10560 gcacgccagc caccaagaac cgcccctgcc agccaccacc cccacccacc ctcccaccac 10620 ccagcctggc tgctccactg tcccgggctg ccctggctgg gggcccgtgc accccggcag 10680 gtggaccagc ctcagccttg gcacctgggc acccagcgga gcggccgccg ctggccacgg 10740 acgagaagat cctcaatggg ctcttctggt atttctcggc ctgcgagaag tgtgtgctgg 10800 cccaggtgtg caaggcctgg cggcgcgtgc tgtaccagcc caagttctgg gcaggcctca 10860 cgccggtgct gcatgccaag gagctctaca acgtgctgcc tggtggcgag aaggagttcg 10920 tgaacctgca gggttttgcc gccagaggct tcgagggctt ctgcctggtt ggcgtctccg 10980 acctggacat ctgtgagttc attgacaact atgcgctctc caagaagggt gtcaaagcca 11040 tgagcctcaa gcgctccacc atcacggacg caggcctcga ggtgtgcacc aggctcgggg 11100 gggacagggc tggggggcag gggcgggcag cagcaaccct atggaggtgg gagcggatgg 11160 tggctttggg gtgggaggaa agaaggactg actacagggg acctcgcgca aagcgaaaat 11220 gtgggtccct tgttcaaaga cactcaagat tttcaagatg gcagcagcag ggcagtggat 11280 taaatgcagg gcccaggccc ggtggctcaa gcctgtcatc ccggcactat gggaggccga 11340 ggcaggccac tcacttgagg ttaggcgttc atgaccagcc tggtcaacat ggtgaaactc 11400 tatttctatt aaaaatgcag aaattagcca ggcctggtgg tgggcacctg tagtcccagc 11460 tacttgggag gacactgagg caggagaatc gcttgaaccc gggaggcggg gggtacagtg 11520 agctgagatc atgccactgc actccagcct gggacctggg caacagagta aggctctgtc 11580 tcaaaatgat tgaatgaatg aatgaataaa taaataaata aataaataca aatgcagtgt 11640 ggggaggatg cgagggggag ctgaggggca gctgggaaag gatcccccag gagtagagg t 11700 cagaggagcc gcctggcagc cgggtctgga gtctaattgt aacgctggtg ccaacgcccc 11760 cagcacctgc cctggccagg ccctgggctc tcgtcccgat gccccgcctc ctcacacact 11820 gccaaatgtt ttcagggttc agctccctgc atgtttacac cctactgctt cctccctgcg 11880 ggctccaccc cttctcctgc aatcctgacc cggcgcccgc ccgcctgcag gttatgcttg 11940 aacagatgca gggcgtggtg cgtctggagc tgtcgggctg caacgacttc accgaggccg 12000 ggctgtggtc cagcctgagc gcgcgcatca cctcgctgag cgtgagtgac tgcatcaacg 12060 tggccgacga cgccatcgcg gccatctcgc agctgctgcc caacctggcg gagctgagcc 12120 tgcaggccta ccacgtgacg gacacggcgc tggcctactt cacggcgcgc cagggccaca 12180 gcacgcacac gctgcgcctg ctctcctgct gggagatcac caaccacggc gtggtcaacg 12240 tggtgcacag cctgcccaac ctcaccgcgc tcagcctctc gggctgctcc aaggtcaccg 12300 acgacggcgt ggagctcgtg gccgagaacc tgcgcaagct gcgcagcctt gacctctcgt 12360 ggtgcccacg catcaccgac atggcgctgg agtacgtggc ctgcgacctg caccgcctag 12420 aggagctcgt gctcgacagg tgcgcgcccc cgggccgcgc cgggcggggc tgggccgggc 12480 ggggctgcac ggggcggggc ggggcggggc tgcacggggc cgggggcggg g gcttccggc 12540 ggggctgggg ctgtggcgcg gcgggcagag ccctcggagc ctggggagtg ggaaccgcag 12600 aaccgcagca cgggttggct ggacccccgt ccttcccggt gggtgggagc gaggaggggc 12660 cagcggctgc tccgggtggg gcaggaaggg attcacgcca ggggcactcc caggaagacg 12720 agggatggcc ggggccaggt gatttgaagg tgggggtccc ctctggtgca accacaggtg 12780 tgtacgcatc acggacactg gcctcagcta tctgtccacc atgtcgtccc tccgcagcct 12840 ctacctgcga tggtgctgcc aggtaccggc tctcacgccc cgggattggg ggatcggcgg 12900 ggaggtgggg gcgcggggag agctcccggc cccactcaga tcctgggaga gtctggggcc 12960 cctccctatc cagcagccga cctaagcgga acgcaggggt cccggcggcc cccacgagca 13020 cttcccagcc gcctcccggc tccggggggg acggaggaac cccgggcttg aacccgccta 13080 gccctccatg gaccccaggg tgccctccgc ctcggtgcga gcggaaatcg ttaagtcgct 13140 cctccactcc cgacaggtgc aagacttcgg gctgaagcac ctcctggccc tggggagttt 13200 gcgcctcctg tctctggcag gtgagacccc cgtttctgct ctgacgctgg cagtgaccac 13260 ccacccccac ttagtccacc cgcccaacct gcccggtcct tgtgcaaact cacacccggc 13320 gcggacacac agtcccgggt ccgaggcgga ggaggaggga ggcg cggccc ggccgtcccc 13380 gcccgagccc tgggccgcgc gcactgagcc gccctctgac cccgccgcag gctgcccgct 13440 gctcaccacc accgggctgt cgggcctggt gcagctgcag gagctggagg agctggagct 13500 gaccaactgc cccggggcca cccccgagct cttcaagtat ttctcgcagc acctgccccg 13560 ctgcctcgtc attgagtagc gcgaggcccc cgccccggtc gcgggaaccc ggccatgacc 13620 tgggcggggg cgcggggcgc cgccgagccc cctcttcccg ccttgcgctc gggggagccc 13680 ccgcgccccc ggcccagcgc gggaggcggg gcgagccgag ggaaagcccc tccccgacct 13740 tcggtccctc cgccctccca gccccgcccc gggcaggggg gcggcgggtg ggcccgcccc 13800 acgcacgcac gcacactcgg ggactttgtg catgcccctc gtgcccgcac tgcacgccgc 13860 cctccgccac gccacagcca cagccgccgc catcactcgc tcgccctccc gcttgggggg 13920 cggggctcgg tccttggggg ggctttgagc tctccagact gtgcccttac cgccttcccc 13980 gccacacccg ctctgtcttc ccactgtccc ccccatcccg ggcagggccc agtgggattg 14040 agggggctgg gtcccccagg acacgggccc agaagagccc cacgggcttc ctgcatcttc 14100 caccgcacca tacctggagc cctccgaggg gtgtcagggg aaacaggcca ccgccaaagc 14160 catggcccgc cgccgagagc ccaggcccca cccgcac ctc ctcacccatc cagcctgacc 14220 cacgcggcct ctcctcctcc ttgccgctgt gtggggcagt cccctgtccg ccccaaaacc 14280 cggccttggt ccctggccag gctgagagaa ttgggcaggg agagggcgga agggctggcg 14340 atcgcttgga gtcattaacg tgatcccagc tgactccggt cggcctcaac ccaggggtgg 14400 cgcaggcacc ttgcaagcct cgagctgtag ccaccctcag gcctgggaag aggcctgggc 14460 cgacctcaca cctcagccct tgcacccggc cgggctcagt tcaggcctgg gcaccgagct 14520 tcaccctggg tgggtctcct caggtggagt ctgcagagtg gacccagcca agggtcaggg 14580 tcagcactgg gtcagcgact ccaatcttcc agtggccagc acaccctaga caccccgagg 14640 agggagggct cctttctagc ctgccccccc acccccactt cacccctccc cagcttccca 14700 aacttctgtc tgcccaaatg ggctctgacc gtgctctgtc ggcccgagac atttggaagt 14760 cctgggggat gctggcaaat ctcagctgtt gctgaggagg gggctgggac cccttcccat 14820 cccaaccttg agccccagga gataccgcgc ccacacccaa tcttgggaca ctccctatct 14880 ggttggaaga gagtaaccag tttccagaga gccagagagt gagagagaga aagagagtga 14940 gagagagaga gaaagagaga gagagatgct gttgaatcag aaacagatca acagcccaaa 15000 gattttcctg tccctggagt gccagcccca ggaagctcca gggctgagtg gtcaggagcc 15060 agtttctcca gcccctcctc cccacaaccc ctagtgggga ggggcagctg tccatttgcc 15120 caaagtatta atgcaactga agctgtgata tttccaacga ctgtaggagg aaaaattaag 15180 gggagagagg aaaacaaaac caaccaaccc ctaaaatcat tttcttattg tacataacga 15240 cctcattctc ctgtatatgc ggaagatata accttatatt tggtaagtgt ttcttgtgct 15300attttatcac gtgacctgtt tataaaaata tatattaaaa aagttgtaaa aaca 15354

Claims (14)

A pharmaceutical composition for inhibiting angiogenesis, comprising the FBXL16 gene or a protein encoded by the gene as an active ingredient. A pharmaceutical composition for preventing or treating metastatic breast cancer, comprising the FBXL16 gene or a protein encoded by the gene as an active ingredient. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits the expression of angiopoietin 1 (Angiopoietin1), angiopoietin2 (Angiopoietin2), PDFG or FGF2. The method according to claim 2, wherein the pharmaceutical composition is characterized in that the inhibition of breast cancer cell migration (migration) and invasion (invasion) ability, the pharmaceutical composition. The pharmaceutical composition of claim 1 or 2, wherein the composition further comprises a pharmaceutically acceptable carrier. The method according to claim 5, wherein the pharmaceutically acceptable carrier is at least one selected from the group consisting of saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, and liposomes, pharmaceutical composition. The pharmaceutical composition of claim 1 or 2, wherein the composition induces degradation of HIF1a. A method for screening a therapeutic agent for metastatic breast cancer, comprising the steps of:
(a) treating cells with a candidate substance in vitro;
(b) measuring the binding level of FBXL16 and HIF1a in the cells; and
(c) selecting a substance that increases the binding level of FBXL16 and HIF1a compared to the candidate substance untreated group. delete delete delete delete delete delete

Images