KR20220010868A - Biomarker for diagnosis or prognosis prediction of metastatic breast cancer and use thereof - Google Patents

Abstract

The present invention relates to a marker for the diagnosis or prognosis of metastatic breast cancer and a use thereof. More specifically, the present invention relates to: a marker composition for the diagnosis or prognosis of metastatic breast cancer comprising an FBXO15 gene or a protein encoded by the gene; a composition for the diagnosis or prognosis of metastatic breast cancer using the same; and a method for providing information for the diagnosis or prognosis of metastatic breast cancer. In the present invention, it has been confirmed that FBXO15 induces ubiquitination and degradation through binding to SOX2, thereby reducing metastatic capacity and stem cell capacity of cancer cells. Accordingly, FBXO15, according to the present invention, can be used as a therapeutic agent for metastatic breast cancer, including a hormone receptor-targeted therapeutic agent.

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 FBXO15 gene or a protein encoded by the gene, and metastatic breast cancer diagnosis or 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 malignant tumor 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 (Korean 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 cells that are negative for a receptor 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 that can treat breast cancer cells. As a result, the E3 ligase FBXO15 showing the greatest expression difference according to the type of breast cancer was selected, and the expression of FBXO15 The present invention was completed by confirming the ability of cancer stem cells 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, which includes the FBXO15 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 FBXO15 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 FBXO15 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 FBXO15 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 FBXO15 gene or a protein encoded by the gene.

In addition, the present invention provides a composition for diagnosing metastatic breast cancer or predicting prognosis, comprising an agent for measuring the mRNA level of the FBXO15 gene or the protein encoded by the 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 FBXO15 gene or a 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 preventing or treating metastatic breast cancer, comprising the FBXO15 gene or a protein encoded by the gene as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition may inhibit the migration and invasion (invasion) of breast cancer cells, or inhibit the stem cell ability of breast cancer stem cells.

In another embodiment of the present invention, the composition may induce degradation of SOX2 at the protein level.

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

The present invention confirmed that FBXO15 induces ubiquitination and degradation through binding to SOX2, thereby reducing metastatic potential and stem cell ability of cancer cells. Accordingly, FBXO15 according to the present invention can be used as a therapeutic agent for metastatic breast cancer including hormone receptor-targeted therapeutics.

Figure 1 shows the expression level of FBXO15 in basal and luminal breast cancers. Figure 1a is the result of confirming the E3 zygote expressed in basal and luminal breast cancers, and Figure 1b is the expression of FBXO15 by breast cancer type through the TCGA database. 1c is the result of confirming the expression of FBXO15 in the luminal breast cancer cell line through the GSE database, FIG. 1d is the result of confirming the breast cancer type through tissue staining, and FIG. 1e is the expression of FBXO15 through Kaplan-Meier analysis The results of confirming the survival rate of patients according to
2 is a result of confirming the invasive ability of FBXO15 and breast cancer cells. FIG. 2a shows the results of confirming the invasive ability through boyden chamber assay when FBXO15 was inhibited using si-RNA in MCF7 luminal breast cancer cells. 2b and 2c show the results of confirming the EMT markers and regulators at the mRNA and protein level, and FIGS. 2d to 2f are FBXO15 using sh-RNA in LM-1 (lung meta-MDAMB231) cells based on the in vitro results. It shows the results of confirming through an in vivo experiment whether metastasis to the lungs when inhibited.
3 is a result of confirming the relationship between FBXO15 and the stem cell ability of breast cancer cells. , Figures 3b and 3c show the results of confirming the mRNA and protein expression levels of SOX2, OCT4 and Nanog, which are major factors regulating stem cell ability, after overexpressing FBXO15 in MDA-MB-231 cells, which are basal breast cancer cells.
Figure 4 confirms the induction of ubiquitination and degradation by the binding of FBXO15 and SOX2, Figures 4a and 4b show that SOX2 and FBXO15 were overexpressed in HEK293T cells and then IP was performed to confirm the binding of the protein, and Figure 4c is MDA -After overexpression of FBXO15 in MB-231 cells, cycloheximide (CHX) was treated to confirm the stabilization of SOX2 protein, and Figure 4d shows ubiquitination assay after overexpression of SOX2 and FBXO15 in HEK293T cells. ) shows the results, and FIG. 4e shows the EMT marker expression changes according to the expression of FBXO15 and SOX2 in MDA-MB-231 cells. A schematic diagram of what to do is shown.

As a result of studying a target that can treat breast cancer cells, the present inventors selected the E3 ligase FBXO15, which shows the greatest expression difference according to the type of breast cancer, and evaluated the cancer stem cell ability and cancer metastasis inhibition ability according to the expression of FBXO15. By confirming, the present invention was completed.

Accordingly, the present invention provides a marker composition for diagnosing or predicting metastatic breast cancer, including the FBXO15 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 mRNA level of the FBXO15 gene or a protein encoded by the gene, and a kit for diagnosing or predicting metastatic breast cancer comprising the same.

In the present invention, the FBXO15 gene may consist of the nucleotide sequence of SEQ ID NO: 1, and homologues of the nucleotide sequence are also included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to the base sequence of SEQ ID NO: 1, respectively. can

In addition, the protein encoded by the FBXO15 gene may consist of the amino acid sequence of SEQ ID NO: 2, and is interpreted to include a sequence showing substantial identity to the sequence shown in SEQ ID NO: 2. The substantial identity is at least 80% when the aligned sequence is analyzed using an algorithm commonly used in the art after aligning the sequence of the present invention with any other sequence as much as possible. 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", 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 the three hormone receptors where metastasis occurs 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 are separated from the primary tumor (hereinafter referred to as "remote site") As a disease that continues to grow from cancer to cancer, the remote site may be, for example, in the same breast (ipsilateral breast) or different breast (contralateral breast) in which 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 may 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 having a length of several bases to several hundreds of bases produced through enzymatic, chemical separation, purification or synthesis. The presence or absence of mRNA can be checked by labeling radioactive isotopes or enzymes, and it 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 contains 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 solution 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.

As 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 FBXO15 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" as used in the present invention provides objective basic information necessary for diagnosis of metastatic breast cancer as a preliminary step for diagnosis, and the clinical judgment or opinion of a doctor is 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 FBXO15 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 FBXO15 by breast cancer type, it was confirmed that the expression of FBXO15 was significantly low in the basal tissue with high metastasis, and the expression of FBXO15 decreased as the breast cancer became malignant (Example 1 and see 2).

In another embodiment of the present invention, as a result of confirming the invasive ability through Boyden chamber assay to analyze the effect of FBXO15 on EMT and metastasis of breast cancer cells, it was confirmed that the invasive ability was increased in the case of si-FBXO15 in which FBXO15 was inhibited. (See Example 3).

In addition, in another embodiment of the present invention, after overexpressing FBXO15 in MDA-MB-231 cells, which are basal breast cancer cells, mRNA and protein expression levels of SOX2, OCT4 and NANOG, which are major factors regulating stem cell ability, were checked, and FBXO15 was overexpressed. As a result of analyzing the association between and cancer stem cells, it was confirmed that the regulation of SOX2 may be directly related to FBXO15 (see Example 4).

Further, in another embodiment of the present invention, after overexpressing SOX and FBXO15 in HEK293T cells, ubiquitination assay was performed. As a result, it was confirmed that ubiquitination increased in the group overexpressing the two proteins. By directly inducing and inhibiting degradation at the level, it was confirmed that cancer stem cell ability and cancer metastasis ability were reduced (see Example 5).

Accordingly, as another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating metastatic breast cancer, comprising the FBXO15 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 FBXO15 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 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 locally applied) 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 route and time of administration, 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, 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 single or multiple. 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, in general 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 FBXO15 can inhibit the penetration and migration ability of breast cancer by inducing the degradation of SOX2 at the protein level. Select a substance that increases the binding of FBXO15 and SOX2 among unknown substances By doing so, it is possible to select a therapeutic agent for metastatic breast cancer.

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 FBXO15 and SOX2 in the cells; and

(c) selecting a substance that increases the binding level of FBXO15 and SOX2 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. E3 ligase selection

In order to confirm the difference in E3 ligase expression according to breast cancer type, six E3 ligase genes (FBXO15, FBXW12, FBXO40, FBXO36, FBXO15, FBXW12, FBXO40, FBXO36, FBXL7 and FBXO16) were confirmed, and FBXO15, which showed the greatest expression difference among the six zygote genes, was selected.

Example 2. Confirmation of differences in expression of FBXO15 according to breast cancer type

First, in order to check whether FBXO15 shows a difference in expression depending on the type of breast cancer, the expression level of FBXO15 was checked using the TCGA and GSE databases for each breast cancer type. 1c, the expression of FBXO15 was high in the luminal breast cancer cell line in the GSE database as shown in FIG. 1c, and from this, it was confirmed that the expression of FBXO15 was high in the luminal type and low in the basal type. did

Next, as a result of staining the breast cancer patient tissue and analyzing the expression level of FBXO15 to directly confirm the database results as described above, as shown in FIG. showed high FBXO15 expression and low FBXO15 expression in basal breast cancer cell lines.

Next, as a result of confirming the correlation between FBXO15 expression and the survival rate of breast cancer patients through Kaplan-Meier survival rate analysis, it was confirmed that the expression of FBXO15 was inversely proportional to the survival rate of the patient as shown in FIG. 1e.

Example 3. Confirmation of invasion ability of breast cancer cells according to FBXO15 expression

To determine whether FBXO15 affects EMT and metastasis of breast cancer cells, si-RNA (Sense: 5'-CGUGAAUUCCUACUCAAAUU-3' (SEQ ID NO: 3), Antisense: 5'-UUUGAGUAGAGAAUUCACGUU- 3') (SEQ ID NO: 4) was used to inhibit FBXO15 and the invasiveness was confirmed through Boyden chamber assay. As shown in FIG. 2a, in the case of si-FBXO15 in which FBXO15 was inhibited, it was confirmed that the invasiveness was increased.

Next, as a result of checking whether FN, VIM, and ZEB1 related to EMT show changes in mRNA and protein levels in FBXO15-inhibited cells using si-RNA, as shown in FIGS. 2b and 2c, control (si-Cont) It was confirmed that both EMT markers and regulators were increased in cells (si-FBXO15) in which FBXO15 was inhibited using si-RNA.

Next, in order to clarify the results of the in vitro experiment, the FBXO15 overexpression vector shown in SEQ ID NO: 5 was used in LM-1 (lung meta-MDAMB231) cells in vivo using xenograft mice as shown in the schematic diagram of FIG. 2D. After making a cell line overexpressing FBXO15, it was injected into a fat pad of a rat and metastasis was analyzed. As a result, as shown in FIGS. 2e and 2f , it was confirmed that metastasis to the lungs was reduced when FBXO15 was overexpressed (LM1-FBXO15) compared to the control group (LM1-Cont).

Example 4. Confirmation of stem cell ability of breast cancer cells according to FBXO15 expression

To determine whether FBXO15 and cancer stem cells are related, FBXO15 was inhibited using si-RNA in luminal breast cancer cells (MCF7) and the increase in sphere size was confirmed. was confirmed to increase.

In addition, the mRNA and protein expression levels of SOX2, OCT4 and NANOG, which are major factors regulating stem cell ability, after overexpressing FBXO15 in MDA-MB-231 cells, which are basal breast cancer cells, were confirmed as shown in FIGS. 3b and 3c. Likewise, although the mRNA level of SOX2 was not changed, only the amount of protein was regulated, which indicates that SOX2 is regulated at the protein level, confirming that the regulation of SOX2 may be directly related to FBXO15.

Example 5. Confirmation of ubiquitination and degradation induction activity according to FBXO15 and SOX2 binding

IP was performed to confirm the association between SOX2 and FBXO15. First, as a result of overexpressing SOX2 and FBXO15 in HEK293T cells, it was confirmed that the two proteins were bound as shown in FIGS. 4A and 4B .

In addition, as a result of confirming the stabilization of SOX2 protein by treatment with Cycloheximide (CHX) after overexpressing FBXO15 in MDA-MB-231 cells, the degradation of SOX2 protein was faster in the group overexpressing FBXO15 as shown in FIG. 4c confirmed to be.

Furthermore, as a result of ubiquitination assay after overexpressing SOX and FBXO15 in HEK293T cells, as shown in FIG. 4d , it was found that ubiquitination increased in the group overexpressing both proteins, MDA-MB-231 As a result of analyzing the expression change of the EMT markers according to the overexpression of FBXO15 and SOX2 in the cells, as shown in FIG. 4e , the EMT marker, which was decreased when only FBXO15 was overexpressed, was increased again when FBXO15 and SOX2 were overexpressed together. Confirmed.

From the above results, as shown in the schematic diagram in FIG. 4f , it was confirmed that FBXO15 reduced cancer stem cell ability and cancer metastasis ability by directly inducing and inhibiting degradation of SOX2 at the protein level.

The above 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> PD20-104 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 1305 <212> DNA <213> FBXO15 <400> 1 atgccttcag aaatcttgct gaagatattt tcctacttgg atgctgtgag ccttctgtgt 60 actggatgtg tgagcaggcg cttttatcat ctagccaatg acaattttat ttggatcgga 120 atctactcaa ctgctttttc acctgcaaga tcaaattgga aatttaattc agtagagaag 180 atagctatgt ctatgagctt tctgtcagtt caggataaag aagctggtta ttggaagaaa 240 gaatatatca caaaacaaat agcatctgta aaagccgcac tagctgacat tctcaaacct 300 gtcaaccctt acacaggcct tccagttaag accaaagagg ccctcagaat atttggttta 360 ggttgggcaa ttatactgaa agaaaaaggt ggaaaagaat atatcatgga gcatgttgat 420 ctttccataa atgacacatc agttactgtt atatggtatg gcaaaaaatg gccatgccta 480 gcatcattgt caaccttaga tttatgtggc atgacaccag tttttaccga ctggtataaa 540 actcccacca aacatagact ccgatggcat tctttaattg caaagtacaa tctgagtcat 600 ttgaccatat ctaccatgat tggctgtgac agactcattc ggatcttctg cctgcaccct 660 ggcctcctgg tgggagtgtg gaagaaggag gaagaactgg cttttgttat ggcaaatctt 720 cattttcatc accttgtgga gaggagcaca ttaggctcgg ctactatccc ctatgaactg 780 cctccacata gccccttttt ggatgatagc cccgagtatg gactgcacgg ctaccaactc 840 catgttgatc tgcacagcgg tggggttttc tacctatgtg gtacatttcg caatctcttc 900 accaagagag gaaatattga aaatggacat gtgaagctca ttgttataca tttaaaaaat 960 aacagagaac acctacctct tattggaaaa gttggcctct cgtggaaaac tgatattttt 1020 gatggctgta taaagagttg ttccatgatg gacgtaactc ttttggatga acatgggaaa 1080 cccttttggt gtttcagttc cccggtgtgc ctgagatcgc ctgccacacc ctctgacagc 1140 tctagcttct tgggacagac atacaacgtg gactacgttg atgcggaagg aagagtgcac 1200 gtggagctgg tgtggatcag agagaccgaa gaatacctta ttgtcaacct ggtcctttat 1260 cttagtatcg caaaaatcaa ccattggttt gggactgaat attag 1305 <210> 2 <211> 434 <212> PRT <213> FBXO15 <400> 2 Met Pro Ser Glu Ile Leu Leu Lys Ile Phe Ser Tyr Leu Asp Ala Val 1 5 10 15 Ser Leu Leu Cys Thr Gly Cys Val Ser Arg Arg Phe Tyr His Leu Ala 20 25 30 Asn Asp Asn Phe Ile Trp Ile Gly Ile Tyr Ser Thr Ala Phe Ser Pro 35 40 45 Ala Arg Ser Asn Trp Lys Phe Asn Ser Val Glu Lys Ile Ala Met Ser 50 55 60 Met Ser Phe Leu Ser Val Gln Asp Lys Glu Ala Gly Tyr Trp Lys Lys 65 70 75 80 Glu Tyr Ile Thr Lys Gln Ile Ala Ser Val Lys Ala Ala Leu Ala Asp 85 90 95 Ile Leu Lys Pro Val Asn Pro Tyr Thr Gly Leu Pro Val Lys Thr Lys 100 105 110 Glu Ala Leu Arg Ile Phe Gly Leu Gly Trp Ala Ile Ile Leu Lys Glu 115 120 125 Lys Gly Gly Lys Glu Tyr Ile Met Glu His Val Asp Leu Ser Ile Asn 130 135 140 Asp Thr Ser Val Thr Val Ile Trp Tyr Gly Lys Lys Trp Pro Cys Leu 145 150 155 160 Ala Ser Leu Ser Thr Leu Asp Leu Cys Gly Met Thr Pro Val Phe Thr 165 170 175 Asp Trp Tyr Lys Thr Pro Thr Lys His Arg Leu Arg Trp His Ser Leu 180 185 190 Ile Ala Lys Tyr Asn Leu Ser His Leu Thr Ile Ser Thr Met Ile Gly 195 200 205 Cys Asp Arg Leu Ile Arg Ile Phe Cys Leu His Pro Gly Leu Leu Val 210 215 220 Gly Val Trp Lys Lys Glu Glu Glu Leu Ala Phe Val Met Ala Asn Leu 225 230 235 240 His Phe His His Leu Val Glu Arg Ser Thr Leu Gly Ser Ala Thr Ile 245 250 255 Pro Tyr Glu Leu Pro Pro His Ser Pro Phe Leu Asp Asp Ser Pro Glu 260 265 270 Tyr Gly Leu His Gly Tyr Gln Leu His Val Asp Leu His Ser Gly Gly 275 280 285 Val Phe Tyr Leu Cys Gly Thr Phe Arg Asn Leu Phe Thr Lys Arg Gly 290 295 300 Asn Ile Glu Asn Gly His Val Lys Leu Ile Val Ile His Leu Lys Asn 305 310 315 320 Asn Arg Glu His Leu Pro Leu Ile Gly Lys Val Gly Leu Ser Trp Lys 325 330 335 Thr Asp Ile Phe Asp Gly Cys Ile Lys Ser Cys Ser Met Met Asp Val 340 345 350 Thr Leu Leu Asp Glu His Gly Lys Pro Phe Trp Cys Phe Ser Ser Pro 355 360 365 Val Cys Leu Arg Ser Pro Ala Thr Pro Ser Asp Ser Ser Ser Phe Leu 370 375 380 Gly Gln Thr Tyr Asn Val Asp Tyr Val Asp Ala Glu Gly Arg Val His 385 390 395 400 Val Glu Leu Val Trp Ile Arg Glu Thr Glu Glu Tyr Leu Ile Val Asn 405 410 415 Leu Val Leu Tyr Leu Ser Ile Ala Lys Ile Asn His Trp Phe Gly Thr 420 425 430 Glu Tyr <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> si-RNA inhibiting FBXO15 expression <400> 3 cgugaauucu cuacucaaau u 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> si-RNA inhibiting FBXO15 expression <400> 4 uuugaguaga gaauucacgu u 21 <210> 5 <211> 1302 <212> DNA <213> Artificial Sequence <220> <223> Vector overexpressing FBXO15 <400> 5 atgccttcag aaatcttgct gaagatattt tcctacttgg atgctgtgag ccttctgtgt 60 actggatgtg tgagcaggcg cttttatcat ctagccaatg acaattttat ttggatcgga 120 atctactcaa ctgctttttc acctgcaaga tcaaattgga aatttaattc agtagagaag 180 atagctatgt ctatgagctt tctgtcagtt caggataaag aagctggtta ttggaagaaa 240 gaatatatca caaaacaaat agcatctgta aaagccgcac tagctgacat tctcaaacct 300 gtcaaccctt acacaggcct tccagttaag accaaagagg ccctcagaat atttggttta 360 ggttgggcaa ttatactgaa agaaaaaggt ggaaaagaat atatcatgga gcatgttgat 420 ctttccataa atgacacatc agttactgtt atatggtatg gcaaaaaatg gccatgccta 480 gcatcattgt caaccttaga tttatgtggc atgacaccag tttttaccga ctggtataaa 540 actcccacca aacatagact ccgatggcat tctttaattg caaagtacaa tctgagtcat 600 ttgaccatat ctaccatgat tggctgtgac agactcattc ggatcttctg cctgcaccct 660 ggcctcctgg tgggagtgtg gaagaaggag gaagaactgg cttttgttat ggcaaatctt 720 cattttcatc accttgtgga gaggagcaca ttaggctcgg ctactatccc ctatgaactg 780 cctccacata gccccttttt ggatgatagc cccgagtatg gactgcacgg ctaccaactc 840 catgttgatc tgcacagcgg tggggttttc tacctatgtg gtacatttcg caatctcttc 900 accaagagag gaaatattga aaatggacat gtgaagctca ttgttataca tttaaaaaat 960 aacagagaac acctacctct tattggaaaa gttggcctct cgtggaaaac tgatattttt 1020 gatggctgta taaagagttg ttccatgatg gacgtaactc ttttggatga acatgggaaa 1080 cccttttggt gtttcagttc cccggtgtgc ctgagatcgc ctgccacacc ctctgacagc 1140 tctagcttct tgggacagac atacaacgtg gactacgttg atgcggaagg aagagtgcac 1200 gtggagctgg tgtggatcag agagaccgaa gaatacctta ttgtcaacct ggtcctttat 1260 cttagtatcg caaaaatcaa ccattggttt gggactgaat at 1302

Claims (12)

A marker composition for diagnosing or predicting metastatic breast cancer, comprising the FBXO15 gene or a protein encoded by the gene. A composition for diagnosing or predicting metastatic breast cancer, comprising an agent for measuring the level of mRNA or protein encoded by the FBXO15 gene. 3. The method of claim 2,
The agent for measuring the mRNA level of the gene is characterized in that the sense and antisense primers or probes that complementarily bind to the mRNA of the gene, the composition. 3. The method of claim 2,
The agent for measuring the protein level is an antibody that specifically binds to the protein encoded by the gene, the composition. A kit for diagnosing metastatic breast cancer or predicting a prognosis, comprising the composition of claim 2. A method for providing information for diagnosing or predicting prognosis of metastatic breast cancer, comprising measuring the expression level of the mRNA of the FBXO15 gene or a protein encoded by the gene in a biological sample derived from a subject. 7. The method of claim 6,
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 (RNase protection assay; RPA), microarray (microarray), characterized in that measured through one or more methods selected from the group consisting of northern blotting (northern blotting), information providing method. 7. The method of claim 6,
The protein expression level was determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, Immunofluorescence (immunofluorescence), ouchterlony (ouchterlony), complement fixation assay (complement fixation assay), characterized in that measured through one or more methods selected from the group consisting of a protein chip (protein chip), How to provide information. A pharmaceutical composition for preventing or treating metastatic breast cancer, comprising the FBXO15 gene or a protein encoded by the gene as an active ingredient. 10. The method of claim 9,
The pharmaceutical composition is characterized in that it inhibits the migration and invasion (invasion) ability of breast cancer cells, or inhibits the stem cell ability of breast cancer stem cells, the pharmaceutical composition. 10. The method of claim 9,
Wherein the composition induces SOX2 degradation at the protein level, a pharmaceutical composition. A method for screening a therapeutic agent for metastatic breast cancer, comprising the steps of:
treating cells with a candidate material in vitro;
measuring the binding level of FBXO15 and SOX2 of the cells; and
Selecting a substance that increases the binding level of FBXO15 and SOX2 compared to the candidate substance untreated group.

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