KR20170119148A - Method for Screening a Composition for Preventing or Treating MYC-AP4 Activated Cancers - Google Patents

Abstract

The present invention relates to a method for screening a composition for preventing or treating cancer, and a method for simultaneous inhibition of AP4 gene and MYC gene expression in vitro.
INDUSTRIAL APPLICABILITY The present invention can be effectively utilized as a reliable anticancer drug screening means by efficiently searching for candidate substances inhibiting the MYC-AP4 axis. The present invention also provides a research tool that can be usefully applied to tumorigenesis mechanism research by providing means for simultaneously inhibiting the expression of MYC and AP4 genes in vitro.

Description

TECHNICAL FIELD The present invention relates to a method for screening a composition for preventing or treating MYC-AP4-activated cancer,

The present invention relates to a method for screening a composition for preventing or treating MYC-AP4 -axis-activated cancer, and a method for simultaneous inhibition of AP4 gene and MYC gene expression in vitro.

The wisdom genetic mechanism regulates gene expression and establishes cell identity. Therefore, the absence of appropriate welfare genetic markers is the cause of diseases involving cancer (1). These welfare genetic markers are recognized by leader proteins that interpret chromatin information and signal chromatin components to promote chromatin remodeling (2,3). The Bromo domain and additional end-domain (BET) family proteins, including the BRD2, BRD3, BRD4 and BRDT (testis-specific) proteins, are not only the most abundant protein modification sites in the cell, Is well known as a leader of acetyllysine residues important for BET regulates various genes involved in cell cycle, cell growth, and inflammation (4). Thus, active research is being conducted on the targeting of such BETs in a variety of therapeutic areas.

MYC, the primary cancer gene, is a transcription factor that includes a basic spiral-ring-helix (bHLH) region that has been studied for over 30 years. The biological mechanisms of MYC have been extensively studied and have addressed various problems. MYC is heterodimerized with the bHLH protein, MAX, and binds to the target gene CA (C / T) GTG. These genes are involved in a wide range of functional spectrum from cell cycle progression and cell growth to epithelial-mesenchymal transition (EMT).

Activating enhancer binding protein 4 (AP4) is thought to be a major mediator of mitogenesis and MYC and EMT mitosis (6, 7). MYC functions as an activator by directly binding to the CACGTG motif in the first intron of AP4 (7). AP4 proteins recognize symmetric DNA sequences such as CAGCTG and form only homodimers, but belong to the bHLH subfamily similar to MYC (7, 8). The roles of the MYC-AP4 axis in cell cycle regulation and tumorigenesis have only recently been discovered (8). Although many studies have reported MYC amplification and / or overexpression in some carcinomas (9), targeting MYC or MYC-AP4 axes remains a challenging challenge.

Recently, a powerful and selective BET Bromo domain small molecule inhibitor JQ1 has been developed. The molecule antagonizes the BET-Bromo domain protein in MYC-dependent transcription in some carcinomas, including multiple myeloma, acute myeloid leukemia, and mixed lineage leukemia 10-12). However, more recent studies have shown that the effect of BET inhibitors is not always dependent on the reduction of MYC (13). Along with the characteristic targeting of epigenetic signaling molecules, BET inhibitors can function differently in a cell context-dependent manner. Therefore, it is important to identify key targets and to identify the underlying mechanisms of BET inhibitors in different cellular environments.

Breast cancer is the most common cancer among women diagnosed with cancer, and is a representative heterogeneous disease that can be classified into several subtypes based on gene expression characteristics and tumor histology (14). Approximately 75% of patients are hormone receptor positive, and treatment has relied on anti-hormone strategies. While most patients respond to endocrine drugs and improve overall survival, many of them eventually become resistant to these drugs (15). It is important to develop a therapeutic strategy to effectively treat hormone receptor-negative breast cancer to prevent such resistance. Recent studies have shown that BET inhibitors are candidates for overcoming resistance to endocrine drugs through the suppression of MYC and P13K signaling (16,17). In the present invention, we examined whether JQ1, an inhibitor of wolfberry genetic leader BRD4, inhibits the MYC-AP4 axis in breast cancer. The present inventors have confirmed that JQ1 inhibits the MYC-AP4 axis in ER-positive and negative breast cancer cell lines. Furthermore, MDA-MB-231, an ER-negative breast cancer cell line that is relatively difficult to target in clinical practice, has been studied. JQ1 down-regulated the MYC-AP4 axis by directly inhibiting the binding of BRD4 to the MYC and AP4 promoters at the initial stage and then induced an anticancer effect including cell cycle arrest, reduced wound healing, and soft agar colonization. The BRD4 loss-of-function experiment further demonstrated that MYC and AP4 are direct targets of BRD4 inhibition. We have found that AP4 loss mimics most anti-tumorigenic effects of JQ1 suggesting that AP4 is a more sensitive target for BRD4-mediated inhibition of MDA-MB-231 cells. Notably, the inventors have for the first time demonstrated that MYC is the downstream target of AP4, and that there is a bi-directional positive loop between MYC and AP4. Thus, MYC-AP4 axis suppression can be further amplified by JQ1. Taken together, the results demonstrated in the present invention demonstrate that BET protein inhibitors are effective against MYC-AP4 axis-activated cancers by targeting various points.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

By identifying the detailed molecular mechanisms of BET (Bromodomain and extra-terminal domain) protein inhibitors, the present inventors have developed a therapeutic strategy for various diseases caused by control disorders of western genetic elements and have developed a search method for highly reliable therapeutic agent candidates I tried my best to do. As a result, JQ1, a small molecule inhibitor of the BET-Bromo domain, inhibits MYC and AP4 axis by directly inhibiting binding of BRD4 to the promoters of MYC and AP4 genes, and found that MYC is a downstream target of AP4, Thereby completing the invention.

Accordingly, an object of the present invention is to provide a screening method of a composition for preventing or treating cancer.

It is another object of the present invention to provide a method for simultaneous inhibition of AP4 gene and MYC gene expression in vitro.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating cancer comprising the steps of:

(a) contacting a test substance with a biological sample comprising an AP4 (activating enhancer binding protein 4) gene and a BRD4 (Bromodomain Containing 4) protein;

(b) measuring the level of binding of the promoter of the AP4 gene and the BRD4 protein, and when the binding is reduced, the test substance is determined as a composition for preventing or treating cancer.

By identifying the detailed molecular mechanisms of BET (Bromodomain and extra-terminal domain) protein inhibitors, the present inventors have developed a therapeutic strategy for various diseases caused by control disorders of western genetic elements and have developed a search method for highly reliable therapeutic agent candidates I tried my best to do. As a result, JQ1, a small molecule inhibitor of the BET-Bromo domain, inhibited the binding of BRD4 to the promoters of MYC and AP4 genes, thereby inhibiting MYC and AP4 axis, and found that MYC is a downstream target of AP4. According to the present invention, a substance that inhibits the binding of BRD4 to a MYC gene promoter, the binding of BRD4 to an AP4 gene promoter and / or the binding of MYC to AP4 has the effect of inhibiting tumors by the same mechanism as JQ1, When the activity is used as a marker, candidates for cancer composition can be screened with high reliability.

As used herein, the term " biological sample " means a biological sample containing an AP4 gene and a BRD4 protein that maintain biological activity, and specifically includes cells, tissues, urine, saliva, blood, plasma But are not limited to, serum samples. In a specific embodiment of the present invention, the biological sample used in the present invention is cancer cells.

More specifically, the cancer cells are MYC-AP4 activated cancer cells, and the cancer to be prevented or treated with the composition screening in the present invention is MYC-AP4 activated cancer. Most specifically, these cancers are selected from the group consisting of breast cancer, liver cancer, colon cancer and esophageal cancer.

The term " test substance " used in reference to the screening method of the present invention refers to the binding between the promoter of the AP4 gene and the BRD4 protein, the binding between the promoter of the MYC gene and the BRD4 protein, or the binding between AP4 and MYC by inhibiting the MYC- ≪ / RTI > is used to screen for < RTI ID = 0.0 > a < / RTI > Such test materials include, but are not limited to, compounds, nucleotides, peptides, and natural extracts. Next, the binding between the promoter of the AP4 gene and the BRD4 protein is measured in the environment in which the test substance is treated. The binding may be measured by a variety of methods known in the art. If the result of the measurement shows that the binding between the promoter of the AP4 gene and the BRD4 protein is significantly reduced, And the like.

As used herein, the term " measurement " encompasses a series of a-priori and deductive processes for deriving an unknown value utilizing specific data, and means the same as " calculation "," prediction ",& . Thus, the term " measurement " in the present invention encompasses both experimental measurements, computational calculations on the in silico , and establishing relationships between a plurality of variables based thereon.

As used herein, the term " decrease binding " means, for example, that the quantification value of the binding level between the promoter of the AP4 gene and the BRD4 protein is significantly lower than that of the control without treatment of the test substance, Means a case where the quantitative value is 5-70% of the control, more specifically, 5-60% of the control.

As used herein, the term " treatment " includes (a) inhibiting the development of a disease, disorder or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition. Therapeutic compositions discovered through the methods of the present invention inhibit the progression, development and proliferation of breast cancer by inhibiting the MYC-AP4 axis and thereby regulate the progression, development and proliferation of breast cancer, It plays a role. Thus, the composition discovered by the method of the present invention may itself be a therapeutic composition for breast cancer, or may be administered together with other pharmacological components and applied as a therapeutic aid for the disease. Herein, the term " treatment " or " therapeutic agent " includes the meaning of " therapeutic aid "

As used herein, the term " prophylactic " means inhibiting the development of a disease or disease in a subject who has never been diagnosed as having a disease or disease, but is likely to suffer from such disease or disease.

The term " administering " or " administering " as used herein refers to the administration of a therapeutically effective amount of a composition of the present invention directly to a subject to form the same amount in the body of the subject. A " therapeutically effective amount " of a composition means an amount of extract sufficient to provide a therapeutic or prophylactic effect to the subject to which the composition is to be administered, including a " prophylactically effective amount ". As used herein, the term " subject " includes without limitation humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the object of the present invention is human.

In a specific embodiment of the present invention, the biological sample used in the present invention further comprises an MYC gene, and the method further comprises the step of measuring the level of binding of the BRD4 protein to the promoter of the MYC gene, The sample is judged to be a composition for preventing or treating cancer.

In a specific embodiment of the present invention, the method further comprises the step of measuring the level of binding between the protein encoded by the AP4 gene and the protein encoded by the MYC gene, wherein when the binding is reduced, Prevention or treatment.

As described above, one embodiment of the present invention is (i) a binding between the promoter of the AP4 gene and BRD4 protein; (Ii) the binding between the promoter of the MYC gene and the BRD4 protein; And (iii) all three indicators of the binding between AP4 and MYC can be screened with high confidence in candidate agents that inhibit tumors with the same mechanisms as JQ1 with antitumor effects in some carcinomas.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating cancer comprising the steps of:

(a) contacting a test substance with a biological sample comprising a compound represented by the following formula (1), an activating enhancer binding protein 4 (AP4) gene, a MYC gene and a BRD4 (Bromodomain Containing 4)

Formula 1

In the general formula, R ₁ to R ₄ are each independently C ₁ -C ₄ alkyl and R ₅ is halogen;

(b) measuring the binding level of the promoter of the AP4 gene and the BRD4 protein or the promoter of the MYC gene and the binding level of the BRD4 protein; when the degree of decrease of the binding is increased, the test substance is a composition for preventing or treating cancer .

Since the biological sample used in the present invention has already been described above, the description thereof is omitted in order to avoid excessive duplication.

According to the present invention, the compound of formula (I) is a reasonable range of derivatives of small molecule JQ1. Since JQ1 inhibits the binding of BRD4 to the promoters of both AP4 and MYC genes, it is believed that the test substance exhibits the same anticancer activity as JQ1 if the test substance suppresses the binding of these substances to the control group more than the control group can do.

As used herein, the term " alkyl " means a straight or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl and the like. C ₁ -C ₄ alkyl means an alkyl group having an alkyl unit having 1 to 4 carbon atoms, and when C ₁ -C ₄ alkyl is substituted, the number of carbon atoms of the substituent is not included. According to a specific embodiment of the present invention, R ₁ to R ₃ in the formula (1) are methyl and R ₄ is t-butyl.

As used herein, the term " halogen " refers to a halogen group element and includes, for example, F, Cl, Br and I. According to a specific embodiment of the present invention, R < ₅ >

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used.

Preferably, the pharmaceutically acceptable salts of the compounds of the present invention are selected from the group consisting of hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, But are not limited to, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, gluturonate, ebonate, glutamate, or aspartate salts, And salts formed using various inorganic acids and organic acids. The compounds of the present invention may also exist in the form of solvates (e.g., hydrates).

According to another aspect of the present invention, there is provided a method for producing an AP4 gene in vitro comprising contacting a biological sample containing an AP4 (activating enhancer binding protein 4) gene and an MYC gene with a compound represented by the following formula Lt; RTI ID = 0.0 > MYC < / RTI > gene expression:

Formula 1

In the general formula, R ₁ to R ₄ are each independently C ₁ -C ₄ alkyl and R ₅ is halogen;

Since the compound of Chemical Formula 1 and the biological sample used in the present invention have already been described above, the description thereof is omitted in order to avoid excessive duplication.

As used herein, the term " expression inhibition " refers to the inhibition of the expression activity that results in the degradation of the gene of interest, and preferably means that the target gene expression is undetectable or meaningless.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a screening method for a composition for preventing or treating cancer, and a method for simultaneous inhibition of AP4 gene and MYC gene expression in vitro.

(b) The present invention can be usefully used as a reliable anticancer screening means by efficiently searching for candidate substances inhibiting the MYC-AP4 axis.

(c) The present invention also provides a research tool that can be usefully applied to the study of tumorigenesis mechanisms by providing means for simultaneously inhibiting the expression of MYC and AP4 genes in vitro .

Figure 1 shows that JQ1 inhibits the MYC-AP4 axis in ER-negative and benign breast cancer cells. MDA-MB-231 (Fig. 1a-1c) and MCF7 (Fig. 1d-1f) cells were treated with increasing concentrations of JQ1 for 24 h. FIGS. 1A and 1D are views showing cell shapes observed at a magnification of × 100. FIG. Figures 1B and 1E show the results of real-time PCR measurement of MYC, AP4 and P21 mRNA levels. Data were expressed as means ± standard error ( ^** P <0.01, ^*** P <0.001 vs. control group). Figures 1c and 1f show the results of Western blotting analysis of MYC, AP4 and P21 protein levels. The results were expressed as representative values of three independent experiments.
Fig. 2 is a graph showing that JQ1 induces antitumor effect. MDA-MB-231 (FIGS. 2A and 2B) and MCF7 (FIG. 2C and 2d) cells were treated with 0.2 μM JQ1 from 6 hours to 24 hours. Figures 2a and 2c show the results of real-time PCR measurement of mRNA levels of MYC, AP4 and P21. Data were expressed as means ± standard error ( ^* P <0.05, ^** P <0.01, ^*** P <0.001 vs. control group). Figures 2b and 2d show the results of Western blotting of MYC, AP4 and P21 protein levels. The results were expressed as representative values of three independent experiments. FIG. 2E shows the result of measurement of DNA content by flow cytometry. Figure 2f shows the results of treatment of MDA-MB-231 cells with 0.2 [mu] M JQ1 and wound healing assay. Figure 2g shows the results of performing soft-ware colony-forming analysis under JQ1 treatment or untreated conditions. The results were expressed as representative values of three independent experiments.
Figure 3 shows that JQ1 directly down-regulates the MYC-AP4 axis by inhibiting the binding of BRD4 to the promoter of both genes. 3A is a schematic diagram of the MYC promoter and the enhancer. Chromatin was immunoprecipitated by binding with anti-BRD4 antibody and binding of BRD4 to MYC regulatory region was analyzed by real-time PCR. Data were expressed as mean ± standard error ( ^*** P <0.001 vs. control). 3B is a schematic diagram of the AP4 promoter and enhancer. Chromatin was immunoprecipitated in association with anti-BRD4 antibody, and binding of BRD4 to AP4 regulatory region was analyzed by real-time PCR. Data were expressed as mean ± standard error ( ^*** P <0.001 vs. control). Figure 3c shows the results of Western blotting of the BRD4 knockdown effect. FIG. 3D shows the results of real-time PCR measurement of mRNA levels of MYC, AP4 and P21 after BRD4 loss. Data were expressed as means ± standard error ( ^* P <0.05, ^** P <0.01, ^*** P <0.001 vs. control).
Figure 4 shows that AP4 is the target of the MYC / MAX dimer, while the MYC / MAX heterozygosity inhibitor 10058-F4 is not as effective as JQ1. MDA-MB-231 cells were treated with increasing concentrations of 10058-F4 for 24 hours. Figure 4A shows the results of real-time PCR of MYC, AP4 and P21 mRNA levels. Data were expressed as mean ± standard error ( ^* P <0.05, ^** P <0.01, ^*** P <0.001 vs. control). FIG. 4B shows the results of analysis of protein levels of MYC, AP4 and P21 by Western blotting. Figure 4c shows the result of observing the cell morphology with a x100 magnification microscope. Figure 4d shows the results of flow cytometry analysis of the DNA content. The results were expressed as representative values of three independent experiments.
FIG. 5 is a graph showing that the knockdown of AP4 reveals a bidirectional positive loop between MYC and AP4, and the base mechanism through inhibition of MYC-AP4 axis has a synergistic effect after JQ1 treatment. Infected AP4-knockdown MDA-MB-231 cells were selected as puromycin. Figure 5A shows the results of real-time PCR of MYC, AP4 and P21 mRNA levels. Data are mean ± standard error ( ^* P <0.05, ^** P <0.01, ^*** P <0.001 vs. control group). FIG. 5B shows the results of analysis of protein levels of MYC, AP4, and P21 by Western blotting. The results shown represent three independent experiments. Figure 5c shows the result of observing the cell morphology with a x100 magnification microscope. The results were expressed as representative values of three independent experiments. FIG. 5D shows the result of measurement of DNA content by flow cytometry. The results were expressed as representative values of three independent experiments. FIG. 5E shows the result of performing soft-colony-forming analysis after softening the AP4 stably. Figure 5f is a schematic diagram of the AP4 binding motif on the MYC promoter. Chromatin was immunoprecipitated with anti-AP4 antibody and the binding of MYC binding motif to AP4 was analyzed by real time PCR after treating JQ1 (0.2 μM) for 24 hours. AP4 combined with MYC with motif was analyzed by real-time PCR after treatment with JQ1 (0.2 μM) for 24 hours. Data were expressed as means ± standard error ( ^*** P <0.001 vs. control). Figure 5g is a picture suggesting a possible mechanism of MYC-AP4 axis inhibition by BRD4 inhibitor JQ1.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

RNA extraction and Reverse transcription PCR

Total RNA was extracted with TRIzol reagent, digested with DNase I, and reverse transcribed using High Capacity cDNA reverse kit (Applied Biosystems). Amplification of the cDNA was done using the LightCycler? 480 Using SYBR Green I Master LightCycler? 480 Ⅱ (above Roche), and the recommended conditions were used. The cDNA was amplified using the following gene-specific primers: RT_MYC, 5'-CCCTGGTGCCGTGAAGC (SEQ ID NO: 1); 3'-TTGCTCGAGTTCTTTCTGCAGA (SEQ ID NO: 2); And RT_AP4, 5'-GCAGGCAATCCAGCACAT (SEQ ID NO: 3); 3'-GGAGGCGGTGTCAGAGGT (SEQ ID NO: 4); And RT_P21, 5'-GAGGCCGGGATGAGTTGGGAGGAG (SEQ ID NO: 5); 3'-CAGCCGGCGTTTGGAGTGGTAGAA (SEQ ID NO: 6) and RT_BRD4, 5'-AAGAAGCGCTTGGAAAACAA (SEQ ID NO: 7); 3'-CAGGTTTTGCTGTCCCTGTT (SEQ ID NO: 8) and RT_P53, 5'-CCCCTCCTGGCCCCTGTCATCTTC (SEQ ID NO: 9); 3'-GCAGCGCCTCACAACCTCCGTCAT (SEQ ID NO: 10); And RT_GAPDH, 5'-GAGTCAACGGATTTGGTCGT (SEQ ID NO: 11); 3'-TGGAAGATGGTGATGGGATT (SEQ ID NO: 12).

Western Blotting

Cells were disrupted in RIPA buffer [150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0) and protease inhibitor] 30% amplitude, 3 seconds). Cell lysates were boiled in Laemmli sample buffer for 3 minutes and SDS-PAGE was performed on 30 μg of each protein. Protein concentrations were determined by Bradford protein analysis. MYC (cat no. 9402S; Cell Signaling Technology or cat no. Ab39688-100; Abcam), AP4 (cat No. HPA001912; Sigma-Aldrich), P21 (cat No. sc-756; Santa Cruz Biotechnology), BRD4 No. 13440), and β-actin (cat no. 49675) (above Cell Signaling Technology). Proteins were transferred to polyvinylidene difluoride membranes and the membranes were blocked for 30 min in TBS buffered TBS buffer containing 0.1% Tween-20 and 5% (w / v) dry skim milk powder for 30 min and the primary antibody : 1,000) overnight. The membrane was then washed with TBS-0.1% Tween-20 and incubated with secondary antibody (dilution 1: 10,000) for 1 hour and then exposed to LAS-3000 image detection system (Fuji) Amersham Life Sciences).

Chromatin Immune sedimentation  analysis( ChIP )

ChIP analysis was performed according to the instructions of Upstate Biotechnology. For each ChIP, 100 μg DNA cut by sonication was removed with protein A magnetic beads and then analyzed with BRD4 (cat no. 13440; Cell Signaling Technology) or AP4 (cat No. HPA001912; Sigma-Aldrich) 40 μg of DNA was precipitated. After immunoprecipitation, the recovered chromatin fragments were subjected to real-time PCR. IgG control experiments were performed on all ChIPs and inserted into IP / Input (1%), and the results were expressed as (IP-IgG) / (Input-IgG). The following primers were used to amplify fragments of chromatin fragments by real-time PCR: the ChIP_MYC promoter, 5'-ACACTAACATCCCACGCTCTG (SEQ ID NO: 13); 3'-GATCAAGAGTCCCAGGGAGA (SEQ ID NO: 14) and ChIP_MYC enhancer 1, 5'-TGCTAATTGTGCCTCTCCTGT (SEQ ID NO: 15); 3'-ACTCCCAGCAAATCAGCCTA (SEQ ID NO: 16); And ChIP_MYC enhancer 2, 5'-GGTCGGACATTCCTGCTTTA (SEQ ID NO: 17); 3'-GAT ATGCGGTCCCTACTCCA (SEQ ID NO: 18) and ChIP_MYC_promoter_AP4 binding motif, 5'-CACTCTCCCTGGGACTCTTG (SEQ ID NO: 19); 3'-GCGCCTACCATTTTCTTTTG (SEQ ID NO: 20) and ChIP_AP4 promoter, 5'-GGGCGCTGCAAATAGTCCTT (SEQ ID NO: 21); 3'-CCGGGCGTGTGTATGTGTGT (SEQ ID NO: 22) and ChIP_AP4 enhancer 1, 5'-CGCGACGTTTGTAAATTGC (SEQ ID NO: 23); 3'-CTCAGATCCCGAGGAAGGA (SEQ ID NO: 24) and ChIP_AP4 enhancer 2, 5'-GAGGTGGGCGTTCTACGG (SEQ ID NO: 25); 3'-GGTTGGGCAGGAGTGTCTAC (SEQ ID NO: 26).

Cell cycle analysis

Cell cycle analysis was performed using the Cycletest Plus DNA reagent kit (BD Biosciences) according to the manufacturer's instructions. The cell cycle characteristics of these cells were analyzed using a FACScan flow cytometer (BD Biosciences).

Soft baby Colony - Soft agar colony-formation assay

Soft agar colony-forming assays were performed in 6-well plates. First, the bottom layer of agar (0.5%) was poured with DMEM (Dulbecco's modified Eagle's medium) (FBS final concentration 10%). After the bottom agar was solidified, MDA-MB-231 cells (1.0 x 10 ⁴ ) were seeded in the top agar (0.3%) with abundant DMEM (FBS final concentration 10%) and incubated at 37 ° C for 21 days. The culture medium was changed 1-2 times a week. The colonies were visualized by staining with 0.005% crystal violet for 1 hour.

Wound - healing analysis

Cells were cultured in confluence for confluency and treated with 0.2 μM JQ1. After blocking the nutrients overnight in serum-free medium, cell monolayers were scraped with a sterile micropipette tip. The initial (0 h) gap width and the remaining gap width at 6 h and 24 h after scarring were measured microscopically.

shRNA  infection

The shBRD4 and shAP4 constructs were purchased from Sigma-Aldrich. Mission lentiviral packaging mix was used for lentivirus production. Infectious mutant cells stably expressing shRNA were selected using 1.25 μg / ml puromycin.

Statistical analysis

The results were expressed as mean ± standard error. Most statistical comparisons were made through a one-way ANOVA followed by a Bonferroni post hoc test using GraphPad Prism. A p value <0.05 was considered statistically significant.

Experiment result

JQ1 In breast cancer cell lines MYC - AP4  Suppress the axis.

ER-negative MDA-MB-231 cells and ER-positive MCF7 cells were used as models to investigate whether JQ1, known as BRD4 inhibitor, targets MYC-AP4 in breast cancer cells. MDA-MB-231 and MCF7 cells were treated with JQ1 at a specific concentration for 24 hours (FIG. 1). As the treatment concentration of JQ1 increased, the morphology of MDA-MB-231 cells became thinner and protruded. In contrast, JQ1 had a minor effect on the morphology of MCF7 cells (Fig. 1d). JQ1 inhibited the MYC-AP4 axis in a dose-dependent manner in MDA-MB-231 and MCF7 cells (Figures 1b, 1c, 1e, and 1f), albeit with different sensitivities. MCF7 cells are more sensitive to inhibition by JQ1 than MDA-MB-231 cells, and this sensitivity may be due to the slightly higher expression of MYC in MDA-MB-231 cells (FIG. 1). P21 expression up-regulation is accompanied by down-regulation of MYC and AP4 (FIGS. 1b, 1c, 1e and 1f), indicating that JQ1 targets the MYC-AP4 axis in both ER-negative and positive breast cancer cell lines.

JQ1 Of breast cancer cells Tumor formation  .

Based on a previous report (11) that acute administration of JQ1 inhibits BRD4, we measured MYC-AP4 axis inhibition by JQ1 (0.2 μM) at early time points in MDA-MB-231 and MCF7 cells. Downregulation of MYC and AP4 was observed 6 h after treatment and accompanied by P21 upregulation (Fig. 2a-2d). In particular, the protein levels of AP4 in both breast cancer cell lines disappeared after 6 hours of treatment (FIGS. 2b and 2d), indicating that AP4 is a very sensitive and direct target of JQ1. In some types of cancer cells, the MYC-AP4-P21 axis is responsible for the G1 arrest (8), thus investigating the effect of JQ1 on the cell cycle. JQ1 treatment increases the percentage of G1-phase cells from 64% to 85% in MDA-MB-231 cells and from 86% to 55% in MCF7 cells (Figure 2e). Next, scratch wound-healing analysis and soft agar colony-forming assay were performed using MDA-MB-231 cells to measure the additional antitumor effect of JQ1. As shown in FIGS. 2f and 2g, JQ1 effectively reduces the wound-healing ability and soft-agar colony formation of MDA-MB-231 cells, indicating that JQ1 exerts an anti-cancer effect on breast cancer cell lines.

JQ1 Lt; RTI ID = 0.0 &gt; promoter BRD4 Lt; RTI ID = 0.0 &gt; MYC - AP4  Adjust the axis spontaneously.

To investigate whether down-regulation of MYC and AP4 by JQ1 is associated with BRD4 binding, we performed immunoprecipitation (ChIP) with MDD-MB-231 using BRD4 antibody (FIGS. 3a and 3b). First, BRD4 (11) which was previously associated with MYC promoter, enhancer 1 and enhancer 2 was measured. As shown in FIG. 3A, it was found that BRD4 binding was initially reduced in the promoter and enhancer 2 when JQ1 was treated, but not decreased in enhancer 1. Next, the previously identified promoters of AP4, BRD4 (7) binding with enhancer 1 and enhancer 2 were measured. The decrease in BRD4 binding was measured mainly in the promoter rather than the enhancer (Fig. 3b), suggesting that inhibition of the MYC-AP4 axis is inhibited by directly inhibiting the binding of BRD4 to the promoter of both genes. To further demonstrate that BRD4 is required for activation of the MYC-AP4 axis, stable BRD4-knockdown cells were constructed and functional loss studies were performed (Figure 3c). After knockdown of BRD4 in MDA-MB-231 cells, the expression of MYC and AP4 was decreased as well as the increase of P21 (Fig. 3d). This indicates that the MYC-AP4 axis of BRA4 It is a direct target.

MYC / MAX heterogeneous Heterozygous  Suppression AP4 But JQ1 as much as Effective  not.

To investigate the activation of the MYC-AP4 axis through the action of the MYC / MAX dimer, a small molecule MYC inhibitor, 10058-F4, was used. 10058-F4 prevents MYC / MAX dimers from binding to the target and inhibits MYC-induced deformation on the targets (18, 19). 10058-F4 does not alter the level of MYC mRNA, while downregulating AP4 mRNA at a dose of 50 μM. This capacity is also sufficient to induce cell morphology changes (Figs. 4A and 4C), suggesting that AP4 is the target of the MYC / MAX dimer. There was a partial decrease in MYC and AP4 protein levels after treatment with 10058-F4 (Fig. 4b), and the effect of 10058-F4 on cell cycle arrest was insignificant compared to the effect of JQ1 (Fig. These results suggest that inhibition of MYC / MAX dimers on the activated MYC-AP4 axis is not as effective as inhibition of BRD4, perhaps due to the smaller down regulation of AP4.

AP4 The knockdown of MYC Wow AP4  Between Bi-directional  Revealing a positive loop, MYC - AP4 The basic mechanism of restraining the axis is JQ1  It shows a synergistic effect after treatment.

Stable AP4-knockdown cell lines were constructed using shRNAs in MDA-MB-231 cells to determine if AP4 is a major target of BRD4 inhibition. MRNA and protein expression of AP4, MYC and P21 were analyzed (Figs. 5A and 5B). As a result, Western blotting demonstrated complete loss of AP4 (Fig. 5b), AP4 loss was shown to inhibit the MYC-AP4 axis and subsequent P21 induction inhibition (Figs. 5a and 5b), cell morphology changes (FIG. 5d) and reduced soft-agar colony formation (FIG. 5e). These results suggest that AP4 is a major target of JQ1 in MDA-MB-231 cells. In addition, we have shown that expression of AP4 is reduced by JQ1 treatment in other cancer cell lines, including cell lines derived from liver, colon and esophagus (data not shown), making AP4 a common target of JQ1 .

Surprisingly, it was observed that when AP4 was knocked down, MYC decreased (FIGS. 5A and 5B). AP4 was known to be a direct target of MYC (6,7); To the best of the present inventors' knowledge, it has not been reported that MYC is a downstream target of AP4. In order to confirm that MYC is a sub-target of AP4 and that there is a bi-directional positive loop between MYC and AP4, ChIP analysis was performed using AP4 antibody after JQ1 treatment. We identified the AP4 binding motif (CAGCTG) in the MTC promoter. We confirmed that AP4 binds to this site, and this binding decreases with the treatment of JQ1 (Figure 5f), indicating that MYC is a sub-target of AP4. Taken together, these data show that MYC-AP4 axis suppression by JQ1 is synergistic by a complex mechanism involving the suppression of BRD4 binding to the MYC and AP4 promoters followed by a secondary inhibition of the bi-directional loop between MYC and AP4 Lt; / RTI &gt;

Additional discussion

The present inventors have confirmed that the inhibitor JQ1 of the western genetic leader BRD4 efficiently targets the bidirectional positive loop between MYC and AP4 to efficiently inhibit the MYC-AP4 axis in breast cancer cells (FIG. 5G). JQ1 inhibits BRD4 binding mainly in the promoter rather than the enhancers of MYC and AP4. The present inventors confirmed that MYC and AP4 are direct targets of BRD4 by producing a stable BRD4-knockdown breast cancer cell line. The inhibitory effect of the MYC / MAX dimerization inhibitor on the activated MYC-AP4 axis was not as effective as the inhibition on BRD4, which appears to modulate AP4 gently downward. AP4 functional loss studies showed that AP4 is an important target for JQ1 and that MYC is a new target for AP4, which is also supported by anti-AP4 ChIP analysis. Downregulation of MYC-AP4 through BRD4 inhibition shows antitumor effect including cell cycle arrest, wound healing reduction and soft agar colony formation. Taken together, our experimental results show that BRD4, a welfare genetic leader, is a key mediator of overexpression of MYC and AP4. This finding provides important clues to the treatment of MYC-AP4 axis-activated cancers.

Inhibitors of the BET family of welfare genetic leaders, including JQ1 and I-BET, have emerged as promising therapeutics for cancer, inflammation and obesity (20). The basic mechanisms of these small molecules often involve the binding of these molecules to the BET protein bromo domain, and this process is completed by lysine acetylation of the histone. Thus, these molecules inhibit the expression of target genes involved in tumorigenesis or inflammatory responses (4, 20). MYC is a primary target in some tumors (11, 12, 21), and a basic mechanism has recently been proposed that involves JQ1 inhibiting MYC by inhibiting super-enhancers (22). Super-enhancers are defined as mass clusters of enhancers that determine cell properties (23,24). Although JQ1 causes a preferential loss of BRD4 in MYC's super-enhancer in multiple myeloma (22), it is unclear whether MYC always has a super-enhancer in a different cellular environment. Indeed, our experimental results show that JQ1 inhibits BRD4 binding to the MYC promoter and enhancer 2, but not the binding of enhancer 1 (Fig. 3a). Therefore, the detailed molecular mechanism of BET protein inhibitors needs further research. AP4 is known to be a MYC-inducible P21 inhibitor (7), but MYC has not been reported as a sub-target of AP4. Thus, the inventors first demonstrated the presence of a bidirectional positive loop between MYC and AP4 (FIG. 5). As shown in FIG. 5, AP4 binds to the MYC promoter. AP4 binding is decreased by JQ1 treatment and AP4 knockdown induces down regulation of MYC. This may be the basic mechanism by which the MYC-AP4 axis inhibition is amplified by BET protein inhibitors. AP4 has also been shown to contribute to several processes, including EMT and metastasis, at the stage of cancer development (6). These results show that JQ1 downregulates AP4 in ER-positive and negative-negative cancer cells when treated at low concentrations at very early stages (FIGS. 1 and 2). These results suggest that AP4 is the most sensitive and important target of BET protein inhibition. AP4 expression in cancer has already been reported to have clinical significance. Increased expression of AP4 is associated with metastatic ability of colorectal cancer (6), and AP4 is an indicator of poor prognosis in non-small cell lung cancer (25). In addition, several studies have reported a key role for AP4 in cancer and immune responses (26-28). Thus, the present inventors' study is expected to contribute to enhance understanding of tumor biology.

It is well known that welfare genetic modifiers and chromatin remodeling control chromatin structure to control gene expression and determine cell characteristics. However, little is known about the basic mechanisms for the effects of specific targets and small molecules targeting these worm genetic elements. These results demonstrate for the first time that BET protein inhibitors inhibit the MYC-AP4 axis by targeting a bidirectional positive loop between MYC and AP4, leading to antitumor effects. Taken together, our experimental results suggest that a better understanding of welfare genetic alterants and chromatin remodeling can facilitate the derivation of new strategies for the treatment of various diseases that arise from abnormalities in welfare genetic elements.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

references

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4. Filippakopoulos P and Knapp S: Targeting bromodomains: Epigenetic readers of lysine acetylation. Nat Rev Drug Discov 13: 337-356, 2014.

5. Meyer N and Penn LZ: Reflecting on 25 years with MYC. Nat Rev Cancer 8: 976-990, 2008.

6. Jackstadt R et al., AP4 is a mediator of epithelial-mesenchymal transition and metastasis in colorectal cancer. J Exp Med 210: 1331-1350, 2013.

7. Jung P et al., AP4 encodes a c-MYC-inducible repressor of p21. Proc Natl Acad Sci USA 105: 15046-15051, 2008.

8. Jung P and Hermeking H: The c-MYC-AP4-p21 cascade. Cell Cycle 8: 982-989, 2009.

9. Dang CV: MYC on the path to cancer. Cell 149: 22-35, 2012.

10. Asangani IA et al., Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 510: 278-282, 2014.

11. Delmore JE et al., BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146: 904-917, 2011.

12. Zuber J et al., RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478: 524-528, 2011.

13. Lockwood WW et al., Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins. Proc Natl Acad Sci USA 109: 19408-19413, 2012.

14. Rakha EA et al., Basal-like breast cancer: A critical review. J Clin Oncol 26: 2568-2581, 2008.

15. Ignatiadis M and Sotiriou C: Luminal breast cancer: From biology to treatment. Nat Rev Clin Oncol 10: 494-506, 2013.

16. Bihani T et al., Resistance to everolimus driven by epigenetic regulation of MYC in ER + breast cancers. Oncotarget 6: 2407-2420, 2015.

17. Stratikopoulos EE et al., Kinase and BET inhibitors together with clamp inhibition of PI3K signaling and overcome resistance to therapy. Cancer Cell 27: 837-851, 2015.

18. Huang MJ et al., A small molecule c-Myc inhibitor, 10058-F4, induction cell-cycle arrest, apoptosis, and myeloid differentiation of human acute myeloid leukemia. Exp Hematol 34: 1480-1489, 2006.

19. Mo H and Henriksson M: Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation. Proc Natl Acad Sci USA 103: 6344-6349, 2006.

20. Belkina AC and Denis GV: BET domain co-regulators in obesity, inflammation and cancer. Nat Rev Cancer 12: 465-477, 2012.

21. Filippakopoulos P et al., Selective inhibition of BET bromodomains. Nature 468: 1067-1073, 2010.

22. Loven J et al., Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153: 320-334, 2013.

23. Hnisz D et al., Super-enhancers in the control of cell identity and disease. Cell 155: 934-947, 2013.

24. Hnisz D et al., Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers. Mol Cell 58: 362-370, 2015.

25. Gong H et al., AP-4 predicts poor prognosis in non small cell lung cancer. Mol Med Rep 10: 336-340, 2014.

26. Chou C et al., C-Myc-induced transcription factor AP4 is required for host mediated by CD8 + T cells. Nat Immunol 15: 884-893, 2014.

27. Jackstadt R and Hinging H: AP4 is required for mitogen- and c-MYC-induced cell cycle progression. Oncotarget 5: 7316-7327, 2014.

28. Jackstadt R, Jung P and Haring H: AP4 directly downregulates p16 and p21 to suppress senescence and mediate transformation. Cell Death Dis 4: e775, 2013.

<110> University-Industry Cooperation Foundation, Konkuk University <120> Method for Screening a Composition for Preventing or Treating          MYC-AP4 Activated Cancers <130> p2016-033 <160> 26 <170> KoPatentin 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> RT-MYC f-primer <400> 1 ccctggtgcc gtgaagc 17 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RT-MYC r-primer <400> 2 ttgctcgagt tctttctgca ga 22 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RT-AP4 f-primer <400> 3 gcaggcaatc cagcacat 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RT-AP4 r-primer <400> 4 ggaggcggtg tcagaggt 18 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> RT-P21 f-primer <400> 5 gaggccggga tgagttggga ggag 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> RT-P21 r-primer <400> 6 cagccggcgt ttggagtggt agaa 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> RT-BRD4 f-primer <400> 7 aagaagcgct tggaaaacaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RT-BRD4 r-primer <400> 8 caggttttgc tgtccctgtt 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RT-P53 f-primer <400> 9 cccctcctgg cccctgtcat cttc 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RT-P53 r-primer <400> 10 gcagcgcctc acaacctccg tcat 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RT-GAPDH f-primer <400> 11 gagtcaacgg atttggtcgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RT-GAPDH r-primer <400> 12 tggaagatgg tgatgggatt 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC promoter f-primer <400> 13 acactaacat cccacgctct g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC promoter r-primer <400> 14 gatcaagagt cccagggaga 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC enhancer 1 f-primer <400> 15 tgctaattgt gcctctcctg t 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC enhancer 1 r-primer <400> 16 actcccagca aatcagccta 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC enhancer 2 f-primer <400> 17 ggtcggacat tcctgcttta 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC enhancer 2 r-primer <400> 18 gatatgcggt ccctactcca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > ChIP-MYC-promoter-AP4 binding motif f-primer <400> 19 cactctccct gggactcttg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-MYC-promoter-AP4 binding motif r-primer <400> 20 gcgcctacca ttttcttttg 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-AP4 promoter f-primer <400> 21 gggcgctgca aatagtcctt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > ChIP-AP4 promoter r-primer <400> 22 ccgggcgtgt gtatgtgtgt 20 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ChIP-AP4 enhancer 1 f-primer <400> 23 cgcgacgttt gtaaattgc 19 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ChIP-AP4 enhancer 1 r-primer <400> 24 ctcagatccc gaggaagga 19 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ChIP-AP4 enhancer 2 f-primer <400> 25 gaggtgggcg ttctacgg 18 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP-AP4 enhancer 2 r-primer <400> 26 ggttgggcag gagtgtctac 20

Claims (18)

A screening method for a composition for preventing or treating cancer comprising the steps of:
(a) contacting a test substance with a biological sample comprising an AP4 (activating enhancer binding protein 4) gene and a BRD4 (Bromodomain Containing 4) protein;
(b) measuring the level of binding of the promoter of the AP4 gene and the BRD4 protein, and when the binding is reduced, the test substance is determined as a composition for preventing or treating cancer.
2. The method of claim 1, wherein the biological sample further comprises an MYC gene, wherein the method further comprises measuring the level of binding of the BRD4 protein to a promoter of the MYC gene, wherein when the binding is decreased, Wherein the composition is determined to be a composition for preventing or treating cancer.
The method according to claim 1, wherein the method further comprises the step of measuring the level of binding between the protein encoded by the AP4 gene and the protein encoded by the MYC gene. When the binding is reduced, &Lt; / RTI &gt;
The method according to claim 1, wherein the biological sample is cancer cells.
5. The method of claim 4, wherein the cancer is an MYC-AP4 activated cancer.
6. The method of claim 5, wherein the MYC-AP4 activated cancer is selected from the group consisting of breast cancer, liver cancer, colon cancer and esophageal cancer.
A screening method for a composition for preventing or treating cancer comprising the steps of:
(a) contacting a test substance with a biological sample comprising a compound represented by the following formula (1), an activating enhancer binding protein 4 (AP4) gene, a MYC gene and a BRD4 (Bromodomain Containing 4)
Formula 1

In the general formula, R ₁ to R ₄ are each independently C ₁ -C ₄ alkyl and R ₅ is halogen;
(b) measuring the binding level of the promoter of the AP4 gene and the BRD4 protein or the promoter of the MYC gene and the binding level of the BRD4 protein; when the degree of decrease of the binding is increased, the test substance is a composition for preventing or treating cancer .
The method according to claim 7, wherein R ₁ to R ₃ in the formula (1) are methyl and R ₄ is t-butyl.
8. The method according to claim 7, wherein R &lt; ₅ &gt;
8. The method of claim 7, wherein the biological sample is cancer cells.

11. The method of claim 10, wherein the cancer is an MYC-AP4 activated cancer.

12. The method of claim 11, wherein the MYC-AP4 activated cancer is selected from the group consisting of breast cancer, liver cancer, colon cancer and esophageal cancer.
A method for simultaneous inhibition of expression of AP4 gene and MYC gene in vitro comprising contacting a biological sample containing AP4 (Activating enhancer binding protein 4) gene and MYC gene with a compound represented by the following formula (1)
Formula 1

In the above general formula, R ₁ to R ₄ are each independently C ₁ -C ₄ alkyl and R ₅ is halogen.
The method according to claim 13, wherein R ₁ to R ₃ in the formula (1) are methyl and R ₄ is t-butyl.
14. The method according to claim 13, wherein R &lt; ₅ &gt;
14. The method according to claim 13, wherein the biological sample is cancer cells.
17. The method of claim 16, wherein the cancer is an MYC-AP4 activated cancer.
18. The method of claim 17, wherein the MYC-AP4 activated cancer is selected from the group consisting of breast cancer, liver cancer, colon cancer, and esophagus cancer.

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