KR101789261B1 - A pharmaceutical composition for treating or preventing breast cancer comprising dj-1 inhibitor as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating breast cancer, which comprises a DJ-1 inhibitor as an active ingredient. The DJ-1 inhibitor according to the present invention has an excellent effect of inhibiting breast cancer by blocking activation of the Notch1 signaling pathway by DJ-1, and thus can be usefully used as a therapeutic agent for breast cancer.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of breast cancer, which contains a DJ-1 inhibitor as an active ingredient. [0002] The present invention relates to a pharmaceutical composition for preventing or treating breast cancer,

The present invention relates to a pharmaceutical composition for preventing or treating breast cancer, which comprises a DJ-1 inhibitor as an active ingredient. The DJ-1 inhibitor according to the present invention inhibits the activation of the Notch1 signaling pathway by DJ-1, down-regulates the interaction of Notch1 and RBP-Jk, or upregulates the interaction of Notch1 and Fbw7, And thus can be usefully used as a therapeutic agent for breast cancer.

Notch is a genetically well-preserved transmembrane protein that plays an important role in embryonic development, cell proliferation, or cell death. Mammals have four Notch genes (Notch1-4) and their ligands Jagged (Jagged 1,2) and Delta (Delta 1,3,4). Notch signaling is expressed upon intercellular contact as a result of the interaction between Notch and its ligand Delta. The Notch receptor is a single pass type I transmembrane protein that normally exists in the cell membrane. When it binds to a ligand, the extracellular domain (ECD) and the intracellular domain (ICD) of the Notch receptor are cleaved, Notch1-IC (Notch intracellular domain), which can move freely in cells, is produced in cells labeled with Notch receptors. When Notch1-IC enters the nucleus, it acts as a transcriptional regulator and affects the expression of several target genes present under the action of Notch. The most important binding proteins of Notch are CSF (CBF1 / recombinant binding protein suppressor of hairless (RBP-Jk)), Drosophila melanogaster Suppressor of Hairless (SuH), Caenorhabditis Lag-1 and mastermind-like (MAML) of elegans . Representative Notch target genes include HES family [Hes / E (spl) family] and HEY family (Hesr / Hey family). In addition to Notch's general target genes such as the HES family and the HEY family, notch-specific protein genes are regulated by c-Myc, CD25, and GATA3.

Recently, Notch has been reported to be associated with many different diseases because it plays an important role in many tissues and organs. Cancer has a high proportion of diseases associated with Notch, and according to certain tissues, Notch is known to be capable of both carcinogenesis and tumor suppressor functions. The association between Notch and tumor was first known in 1991 by chromosome translocation of Notch in T-cell acute lymphoblastic leukemias / lymphomas (TALL). This chromosomal translocation results in the presence of Notch1 in a continuously activated state, thereby altering the expression of transcriptional regulatory factors and inducing TALL. Since then, Notch1 has been shown to play a role in the normal development of leukemia as well as of T cell progenitors and the precise formation of T-cell receptors. In addition to blood cancers such as leukemia, Notch plays an important role in many solid tumors such as breast cancer, adenocarcinoma, colorectal cancer, lung cancer and melanoma. Notch's oncogene is known to play a role as a tumor suppressor in blood cancers such as B-cell acute lymphoblastic leukemia (B-ALL) as well as solid tumors such as the role and spontaneous basal cell carcinomas. Thus, Notch is emerging as one of many important goals that can provide clues and suggestions for treating various human diseases.

Control of Notch signaling results in several steps such as ligand and receptor expression patterns, Notch-ligand interactions, receptor and ligand trafficking, and covalent modifications such as glycosylation, phosphorylation, acetylation, nitrification and ubiquitination . Notch's E3 ligase plays an important role in determining the turnover rate of Notch signaling, since almost all of the Notch signaling occurs through ubiquitination. Five E3 ligases regulate Notch, Notch ligand, or Notch antagonist. LNX is a positive regulator of the Notch antagonist Numb, a membrane-associated protein that inhibits the function of Notch receptors. Neuralized and Mindbomb activate the ligand through the mono-ubiquitination and endocytosis of the Notch ligand Delta. E3 ligase Itch binds to the intracellular domain of Notch via its four WW motifs and acts through the Notch extracellular domain cleavage form to activate ubiquitination of Notch1-IC through its HECT domain. In Notch signaling, the most important protein in the half-life of Notch's intracellular domain is Fbw7. Fbw7 binds to Notch1-IC through its WD40 domain and regulates its ubiquitination and degradation through the proteasome system, resulting in PEST (proline-, glutamate-, serine-, threonine-rich) domain-dependent Notch intracellular degradation Activate.

Fbw7 / Cdc4 (F-box and WD40 repeat domain-containing 7) are tumor suppressor proteins that regulate various cellular processes such as cell proliferation, cell-cycle progression, and are often inactivated in cancer. Mutations in Fbw7 lead to a variety of human cancers, such as breast cancer, through gene mutation and down-regulation of expression. Fbw7 is an F-box protein that regulates the recognition and binding of substrate proteins by binding a substrate adapter for SCF ^FBW7 ( ^complex of Skp1, CUL1 and F-box proteins) ubiquitin ligase complex. SCF ^FBW7 has been shown to ^inhibit cell growth, proliferation, differentiation and survival, such as c-Myc, cyclin E, Mcl-1, c-Jun, mammalian target of rapamycin (mTOR) The proto-oncogene is degraded. Surprisingly, most Fbw7 substrates are known to act as oncogene in a variety of human cancers.

DJ-1, also called PARK7, is known to be involved in Parkinson's disease (PD). The functional inactivation of DJ-1 is caused by a mutation of Leu166 to Pro which blocks its stabilization. Other pathogenic mutations M26I, E64D, L10P and P158Δ have also been reported to cause early-onset Parkinson's disease with autosomal recessive inheritance. Functionally, DJ-1 has scavenging activity against oxidative stress by self-oxidation of its cysteine residues. A highly conserved Cys106 residue is essential for its oxidation. DJ-1 behaves as lys130 under nitrosative stress (NS). DJ-1 is involved in protection against oxidative stress-induced cell death.

Although DJ-1 is known primarily for its neuroprotective function in Parkinson's disease, DJ-1 was first identified as a carcinogenic gene with H-ras in NIH3T3 cells with a transformation activity. Recent studies have shown that DJ-1 is closely related to tumorigenesis of malignant tumors. The level of expression of DJ-I is increased in a variety of cancers including breast, pancreatic, lung and non-small cell lung cancer. It also indicates that DJ-1 is involved in cell proliferation and survival mechanisms, typically the PI3K / Akt / mTOR pathway. DJ-1 promotes AKT phosphorylation, which protects cells from stress-induced apoptosis. DJ-1 modulates the PI3K survival pathway by inhibiting the function of PTEN, a tumor suppressor gene. DJ-1 positively regulates the HIF1 transcriptional activity promoted by the PI3K / Akt / mTOR pathway. Under hypoxic conditions, DJ-1 increases resistance to hypoxic stress by HIF1 induction. DJ-1 also regulates the activity of the metabolic sensor AMPK during hypoxia. In another aspect, DJ-1 also regulates cell cycle arrest mechanism via a p53-dependent pathway. The loss of p53 correlates with the high expression of DJ-1, and DJ-1 is involved in p53-mediated cellular responses to oxidative stress. DJ-1 is known to induce survivin expression which inhibits apoptosis and blocks tumor cell division and angiogenesis.

The present inventors have found that DJ-1 is involved in breast cancer by activating the Notch1 signaling pathway. Furthermore, we have shown that DJ-1 promotes the formation of Notch1 transcription complexes with RBP-JK, while it interacts with Fbw7, the Notch1 E3 ligase and Pin1, resulting in Fbw7 self-ubiquitination, thereby enhancing the stability of Notch1 protein Respectively. Therefore, the present inventors intend to provide the use of a DJ-I inhibitor in the treatment or prevention of breast cancer.

One. DJ -1 in breast cancer cells Notch1  Signal pathway Promoting  Identification

We first assessed the protein levels of DJ-1, Notch1 and its target genes Hes1 and Hes5 in human breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-468 and SK-BR-3. As a result, expression of DJ-1, Notch1 and its target genes Hes1 and Hes5 in breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-468 and SK- Level was upregulated (Figure 1A). In addition, in order to examine whether DJ-1 and Notch1 are involved in breast cancer, it was examined whether the viability of MCF-7 cells was regulated by DJ-1 or Notch1 expression inhibition. As a result, it was confirmed that when MCF-7 cells were treated with DAPT known as gamma-secretase inhibitor, cell viability was suppressed (FIG. 1B). Surprisingly, inhibition of cell viability by DAPT was more effective in MCF-7 cells with DJ-1 removed using specific shRNAs.

The present inventors analyzed the transcriptional activity of the Notch1 target gene according to the DJ-1 expression level in order to examine whether DJ-1 regulates the Notch1 signaling pathway and affects cell viability. Luciferase reporter assay was performed on HEK293 cells using Hes1-Luc, Hes5-Luc and artificial 4xCSL-Luc as the luciferase reporter gene. As a result, it was confirmed that Notch1 transcriptional activity was promoted as the dose of DJ-1 was increased (Fig. 2A-C).

We also examined whether Notch1-mediated gene activity changes in the absence of DJ-1 in order to examine the role of DJ-1 in upregulating Notch1 transcriptional activity. The transcriptional induction of the Notch1-dependent gene was measured by real-time quantitative PCR (Q-PCR) using total RNA isolated from MCF-7 cells stably expressing the control shRNA and DJ-1 shRNA. It was confirmed that knockdown downregulated the mRNA levels of Hes1 and Hes5. Surprisingly, the Notch1 target gene expression inhibitory effect of shDJ-1 is not limited to the Hes family gene, and the mRNA levels of other Notch1 target genes such as Hes6, Hey1, Hey2, p21, p27 and c-Myc are also affected by DJ-1 knockdown (Fig. 3). Thus, it has been demonstrated that overexpression of DJ-I promotes the activity of Notch1-mediated gene, leading to cell growth of tumor cells.

2. DJ -1 is Notch1 - IC  And RBP - Jk Interacting with Notch1  Identification of involvement in transcription complex formation

The present inventors in order to investigate the mechanism by Notch1 signaling pathway DJ-1 using the purified His-DJ-1 protein fixed on nickel agarose beads vitro binding assay was performed. As a result, it was confirmed that DJ-1 directly binds to Notch1 (Fig. 4). In the nucleus, Notch1-IC interacts with RBP-Jk recruited to DNA to regulate target gene expression. RBP-Jk can act as a transcription repressor or activator depending on the recruiting protein complex. Accordingly, the present inventors analyzed the correlation between DJ-1 and RBP-Jk complexes in the same manner that DJ-1 induces Notch1 transcription activity. As a result, DJ-1 interacted with RBP-Jk through its c-terminal domain (Fig. 5A-C), and DJ-1 simultaneously interacted with Notch1 and RBP- Jk to form a ternary complex (Fig. 6A). Indeed, the triple complex was also detected in the MDA-MB-231 breast cancer cell line (Fig. 6B).

We conducted an endogenous immunoprecipitation assay with or without DJ-1 in the breast cancer cell line MCF-7 to examine the regulation function of DJ-1 in the Notch1 transcription complex. As a result, it was confirmed that the elimination of DJ-1 reduced the interaction between Notch1 and RBP-Jk (FIG. 7A), but the inhibitor of Notch1 transcription complex, SMRT, was more strongly bound to RBP-Jk (FIG. Thus, DJ-1 has been shown to interact with Notch1 transcription complex formation.

3. DJ -1 is Fbw7 E3 Ligase  through Notch1 - IC  Identifying the stability of the protein

We confirmed the half-life of the Notch1 protein through cyclohexamide (CHX) tracing since DJ-1 knockdown using specific shRNAs gradually decreases Notch1 protein levels (Fig. 8A). As a result, the half-life of Notch1 protein was shortened when DJ-1 shRN was used compared to the shRNA control (Fig. 8B), but it was confirmed that the mRNA level of Notch1 was not regulated by DJ-1 knockdown (Fig. 8C). Thus, the present inventors have anticipated that DJ-1 is directly involved in the stability of the Notch1 protein. Notch1-IC is degraded into ubiquitin-proteasome system by F-box protein Fbw7 / Sel-10 / hCdc4 / Ago. We therefore examined whether DJ-1 influences Fbw7-mediated Notch1 degradation to see if Fbw7 acts as a mediator of down-regulation of Notch1-IC by DJ-1. As a result of the immunoprecipitation assay, it was confirmed that DJ-1 significantly inhibited the interaction between Notch1 and Fbw7 (FIG. 9A). In HEK293 cells transformed with DJ-1 shRNA, the endogenous binding between Notch1-IC and Fbw7 was found to be greater than in cells transfected with control shRNA (FIG. 9B). This demonstrates that DJ-1 inhibits the interaction of Notch1-IC and Fbw7 in normal cells and can positively regulate transactivity of the Notch1-IC target gene.

4. DJ -1 is Pin1 with Fbw7 of Ubiquitination Promoting  Identification

Fbw7 is known to be a tumor suppressor involved in the regulation of cell pathway by promoting the degradation of oncogene including Notch1. Interestingly, we have found that Fbw7 protein levels are inversely correlated with DJ-I. Fbw7 protein levels were reduced by DJ-1 overexpression, whereas DJ-1 knockdown increased Fbw7 protein levels compared to control. This with the assumption that the inventors DJ-1 directly controls the Fbw7 the basis, in order to verify this vitro binding assay and immunoprecipitation assay were performed. in As a result of the in vitro binding assay, the interaction between His-DJ-1 and Fbw7 was detected in the bead complex (Fig. 10A). As a result of the immunoprecipitation assay, transformation with a vector encoding Myc-Fbw7 and Flag-DJ- Lt; RTI ID = 0.0 > HEK293 < / RTI > In addition, endogenous binding between DJ-1 and Fbw7 was confirmed (FIG. 10C). The inventors then examined whether DJ-1 modulates Fbw7 protein levels and shortens the half-life of Fbw7. Surprisingly, it was confirmed that DJ-1 expression increase in HEK293 cells caused instability of Fbw7 whereas DJ-1 expression inhibition increased Fbw7 stability (Fig. 10D). The half-life of the endogenous Fbw7 protein was about 4 hours, but increased up to about 8 hours when DJ-1-specific shRNA was introduced (Fig. 10E). It has thus been demonstrated that DJ-1 physically interacts with Fbw7 to modulate the stability of the Fbw7 protein.

Pin1 is known to interact with Fbw7 in a phosphorylation-dependent manner to promote protein degradation by Fbw7 self-ubiquitination and Fbw7 dimer formation disturbances. Therefore, the present inventors tested whether the regulation of Fbw7 by DJ-1 is mediated by Pin1 in transformed HEK293 cells. Significantly, the loss of Pin1 by shPin1 disrupted the effect of DJ-1 on Fbw7 levels (Fig. 11A). Surprisingly, induction of Pin1 shRNA in HEK293 cells destabilized DJ-1 expression. Indeed, induction of Pin1 expression gradually decreased the level of Fbw7, while increased the level of DJ-1 protein (FIG. 11B). In addition, the present inventors have examined whether DJ-I knockdown affects Pin1-induced Fbw7 destabilization. Pin1 reduced Fbw7 protein levels in control HEK293 cells, but not in DJ-1 deficient conditions, indicating that Fbw7 destabilization by Pin1 is also dependent on DJ-1 expression (Fig. 12A). In addition, while DJ-1 promotes interaction between Fbw7 and Pin1 (Figure 12B), DJ-1 and Pin1 inhibit dimer formation preventing self-ubiquitination of Fbw7 (Figure 12C) . ≪ / RTI > Based on this, we examined whether DJ-1 is involved in the self-ubiquitination of Fbw7, and HEK293 cells were transformed with Flag-Fbw7-encoding vector and His-DJ-1 or DJ-1 shRNA The ubiquitination assay with HA-ubiquitin was performed. Immunoblotting analysis confirmed that DJ-1 knockdown inhibited ubiquitination of Fbw7 while DJ-1 overexpression promoted ubiquitination of Fbw7 (FIG. 13A). To confirm the importance of the Pin1 mediator, repeated experiments with Pin1 wild type and T205A mutations and Pin1-binding deficient mutations confirmed that the Fbw7 T205A mutation inhibited DJ-1 induced ubiquitination of Fbw7 (FIG. 13B). Fbw7 ubiquitination was significantly increased in cells overexpressing both DJ-1 and Pin1 (Fig. 13C). It has thus been demonstrated that DJ-1 prevents Fbw7-mediated Notch1 degradation by promoting self-ubiquitination of Fbw7.

5. DJ -1 is Fbw7  In a dependent way breast cancer Tumor formation Promoting  Identification

To determine whether DJ-1 inducible tumor formation is mediated by Fbw7, we constructed MCF-7 cells in which the depletion state of DJ-1 and Fbw7 was maintained using an shRNA vector. The DJ-1 shRNA-expressing cells showed a decrease in cell proliferation rate compared to the control shRNA cells, but the cells co-expressing Fbw7 shRNA and DJ-1 shRNA showed a restored cell proliferation rate (FIG. 14). In addition, the present inventors examined cell migration activity through the wound healing assay. Referring to FIG. 15A, it was confirmed that the depletion of DJ-1 significantly reduced the cell migration ability, but the cell migration ability was restored by Fbw7 depletion. In addition, the present inventors confirmed similar results through the transwell movement assay (FIG. 15B) and the Martrig cell invasion assay (FIG. 15C). The knockdown of DJ-1 reduced the number of lesions and colonies in soft agar, but knockdown of both DJ-1 and Fbw7 restored colony formation (Fig. 16A-B).

To study the biological function of DJ-1 and to establish a mouse transplantation model, MCF-7 tumor cells stably expressing DJ-1 shRNA and Fbw7 shRNA were subcutaneously injected into nude mice and tumor growth (Fig. 17). Similar to the in vitro growth curve, growth of tumor cells was inhibited in endogenous DJ-1-depleted breast cancer transplantation model, but this inhibitory effect was confirmed to be nullified by Fbw7 collapse.

The present inventors finally examined the expression levels of DJ-1, Notch1 and Fbw7 in human breast cancer tissues (Fig. 18) in order to confirm the clinical relevance of the experimental data. A total of 43 human tumor tissue samples were immunoblotted and compared with normal breast tissue. DJ-1 was overexpressed in 41/43 (95%) samples compared to normal breast tissue, and Notch1 was promoted in 31/43 (72%) samples. On the other hand, the level of Fbw7 was higher than that of normal tissue in 36/43 (84%) samples. Thus, DJ-1 showed a correlation with Notch1 but was inversely correlated with Fbw7 (Fig. 19). It has thus been demonstrated that DJ-1 significantly promotes the growth of breast cancer by modulating migration and colony forming activity through the Fbw7 mediated pathway.

According to one embodiment, there is provided a pharmaceutical composition for the prevention or treatment of breast cancer comprising an siRNA, shRNA or antisense nucleic acid specific to the DJ-1 gene which is an expression inhibitor of the DJ-1 gene.

The pharmaceutical composition for preventing or treating breast cancer according to the present invention is characterized in that the DJ-1 is composed of the amino acid sequence of SEQ ID NO: 1.

In the pharmaceutical composition for preventing or treating breast cancer according to the present invention, the expression inhibitor of DJ-1 gene is characterized in that the expression level of the Notch1 target gene comprising Notch1 or Hes1 and Hes5 is down-regulated.

The pharmaceutical composition for preventing or treating breast cancer according to the present invention is characterized in that the expression inhibitor of DJ-1 gene down-regulates the interaction between Notch1 and RBP-Jk.

In the pharmaceutical composition for preventing or treating breast cancer according to the present invention, the DJ-1 gene expression inhibitor is characterized in that the interaction between Notch1 and Fbw7 is upregulated.

According to another embodiment, there is provided a pharmaceutical composition for the prevention or treatment of breast cancer comprising an antibody specific for the DJ-1 protein, which is an activity inhibitor of the DJ-1 protein.

The pharmaceutical composition for preventing or treating breast cancer according to the present invention is characterized in that the DJ-1 is composed of the amino acid sequence of SEQ ID NO: 1.

In the pharmaceutical composition for preventing or treating breast cancer according to the present invention, the activity inhibitor of the DJ-1 protein is characterized by down-regulating the activity of the Notch1 target protein comprising Notch1 or Hes1 and Hes5.

In the pharmaceutical composition for preventing or treating breast cancer according to the present invention, the activity inhibitor of DJ-1 protein is characterized by down-regulating the interaction between Notch1 and RBP-Jk.

In the pharmaceutical composition for preventing or treating breast cancer according to the present invention, the inhibitor of the activity of the DJ-1 protein is characterized in that the interaction between Notch1 and Fbw7 is upregulated.

According to another embodiment,

(a) treating the test substance with an expression cell line;

(b) measuring the level of expression or activity of the DJ-1 protein in the cell line; And

(c) selecting a test substance whose expression or activity level of the DJ-1 protein is decreased as compared with a control group in which the test substance is not treated, is provided.

In the method for screening a preventive or therapeutic agent for breast cancer according to the present invention, the expression of the DJ-1 protein in the step (b) may be determined by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA) Is characterized by histochemical, RT-PCR, Western blotting or flow cytometry (FACS).

In the method for screening a preventive or therapeutic agent for breast cancer according to the present invention, the activity of the DJ-1 protein in the step (b) may be measured by SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry or protein chip .

The DJ-1 inhibitor according to the present invention has an excellent effect of inhibiting breast cancer by blocking activation of the Notch1 signaling pathway by DJ-1, and thus can be usefully used as a therapeutic agent for breast cancer.

Fig. 1 (A) is a graph showing the effect of the MCF-10A normal breast cell line and DJ-1, Notchl and its target gene on breast cancer cell lines including MCF7, MDA-MB-231, MDA-MB-468 and SK- The results of Western blotting detection are shown. β-actin was used as a loading control. FIG. 1 (B) shows the results of the trypan blue exclusion assay using extracts prepared from parent MCF-7 cells or DJ-1 stably knocked down MCF-7 induced cells.
Figure 2 (AC) shows the luciferase activity of HEK293 cells transfected with expression vectors for Notch1-IC, DJ-1, 4xCSL, Hes1 or Hes5-Luc, β-galactosidase.
FIG. 3 shows a primer for detecting a Notch1 target gene comprising Hes1, Hes5, Hes6, Hey1, Hey2, p21, p27 and c-Myc in MCF-7 cells stably expressing shDJ-1 and control MCF- Real-time q-PCR results are shown.
Figure 4 shows pull-down assay results for immobilized His-DJ-1 and HEK293 cells.
Figure 5 (A) shows pull-down assay results for immobilized His-DJ-1 and HEK293 cells. Figure 5 (B) shows the immunoprecipitation assay results of transfected HEK293 cells. Figure 5 (C) shows Western blotting results of anti-DJ-1 immunoreactive precipitate in DJ-1 knockdown HEK293 cells and control.
6 (A) shows the results of immunoprecipitation assay in HEK293 cells transfected with an expression vector. Figure 6 (B) shows the immunoprecipitation assay of endogenous protein levels in MDA-MB-231 breast cancer cell lines.
FIG. 7 (AB) shows the results of the immunoprecipitation assay in the presence and absence of DJ-1 in the MCF-7 breast cancer cell line.
8 (A) shows Western blotting analysis results on Notch1-IC protein stability according to DJ-1 expression. Figure 8 (B) shows Western blotting analysis results for Notch1-IC protein half-life in HEK293 cells treated with 100 [mu] M cycloheximide (CHX) over time. β-actin was used as a loading control. Fig. 8 (C) shows the results of real-time q-PCR analysis of Notch1 mRNA expression in transfected HEK293 cells, respectively.
Figure 9 (A) shows the immunoprecipitation assay results in HEK293 cells transfected with the indicated expression vectors. FIG. 9 (B) shows immuno-precipitation assay results for endogenous protein levels according to the presence of DJ-1 in HEK293 cells.
Figure 10 (AC) shows the results of immunoprecipitation assay using HEK293 cell extract transfected with the indicated constructs. 10 (D) shows Western blotting analysis results for Notch1-IC protein stability in HEK293 cells transfected with Flag-DJ-1 or shDJ-1. Figure 10 (E) shows Western blotting analysis results after treatment with cycloheximide (CHX) for the indicated time in DJ-1 depleted and control HEK293 cells.
Figure 11 (AB) shows the Western blotting assay results for HEK293 cells transfected with the indicated constructs.
Figure 12 (A) shows the Western blotting analysis results for HEK293 cells transfected with the indicated constructs. 12 (BC) shows the result of Western blotting analysis using a specific antibody in HEK293 cells expressing the expressed protein.
Figure 13 (AC) shows the results of the ubiquitination assay of HEK293 cells expressing the constructs after addition of 5 [mu] M MG132 for 6 hours.
Figure 14 shows the results of weighing tumors in Balb / c-nude mice for MDA-MB-231 SQ cells stably expressing DJ-1 and Fbw7 shRNAs.
Figure 15 (A) shows the wound-healing assay results for MDA-MB-231 cells transfected with DJ-1 and Fbw7 specific shRNAs. Fig. 15 (B) shows the results of a transwell mobility assay of MCF7 cells stably expressing shDJ-1 and shFbw7. 15 (C) shows the results of Matrigel invasion assay using MCF7 cells stably expressing shDJ-1 and shFbw7.
16 (A) shows the result of colony formation and soft agar assay using MCF7 cells stably expressing shDJ-1 and shFbw7. Figure 16 (B) shows the results of colony formation assay on MCF7 cells expressing shDJ-1 and shFbw7.
Figure 17 shows the results of xenograft tumor formation assays in Balb / c-nude mice for MDA-MB-231 SQ cells expressing shRNAs of DJ-1 and Fbw7.
18 shows immunoblot analysis results for anti-DJ-1, anti-Notch1 and anti-Fbw7 antibodies in human breast cancer tissue samples.
Figure 19 shows the statistical analysis of the ratio of expression levels of DJ-1, Notch1, and Fbw7 in 42 human breast cancer tissues compared to the standard test calculated using the paired t-test.

I. General Technology

Unless otherwise indicated, the methods and techniques of the present invention will generally be described in accordance with conventional methods well known in the art and, unless otherwise indicated, in various general and specific references cited and discussed throughout this specification As shown in FIG.

II. Pharmaceutical composition for prevention or treatment of breast cancer

The present invention provides a pharmaceutical composition for preventing or treating breast cancer, which comprises an agent for inhibiting expression of a DJ-1 gene or an agent for inhibiting the activity of a DJ-1 protein.

The term "DJ-1 ", also referred to herein as" PARK7 ", is also known as the autosomal recessive gene of Parkinson's disease. DJ-1 is a homodimer belonging to the Thi / Pfp1 superfamily and is well conserved from human to E. coli and is distributed in the cytoplasm, nucleus and mitochondria. The DJ-1 protein performs various functions such as antioxidant activity, chaperone-like properties and transcriptional regulation.

As used herein, the term "agent for inhibiting the expression of a DJ-1 gene" is a substance that inhibits the expression of a gene level of DJ-1, preferably an siRNA, shRNA or antisense nucleic acid that specifically binds to the DJ- .

The term "siRNA" as used herein means a short double-stranded RNA capable of inducing RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene, and an antisense RNA strand having a complementary sequence. Since siRNA can inhibit the expression of a target gene, it can be provided as an efficient gene knockdown method or gene therapy method. The siRNA is not limited to the complete pairing of the double-stranded RNA portions that are mated with each other, but the siRNA may be paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one of the chains) And a portion that is not achieved may be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The adhesive end structure can be a structure having a 3 'end protruding structure and a 5' end protruding structure. The number of protruding bases is not limited. The siRNA may be a small RNA (for example, a natural RNA molecule such as a tRNA, a rRNA, or a viral RNA, or an artificial RNA molecule) at a protruding portion at one end within a range capable of maintaining the effect of suppressing the expression of a target gene, . ≪ / RTI > The siRNA end structure does not need to have a truncation structure on both sides, and may be a step-loop structure in which the terminal region of the double-stranded RNA is connected by linker RNA. The siRNA according to the present invention may be a complete form having polynucleotide pairing itself, that is, a form that is directly introduced into a cell through two transformation processes in which an siRNA is directly synthesized in vitro, Single-stranded oligonucleotide fragments and their reverse-phase trefoil can be derived from single-stranded polynucleotides separated by a spacer, for example, siRNA expression vectors or siRNA expression vectors or PCR-derived siRNAs The expression cassette may be introduced into the cell through a transformation or infection process. The determination of how to prepare siRNAs and introduce them into cells or animals may depend on the objective and the cellular biological function of the target gene product.

As used herein, the term "shRNA" is intended to overcome the disadvantages of high cost biosynthetic cost of siRNA, short term maintenance of RNA interference effect due to low cell transfection efficiency, And a plasmid expression vector system. The shRNA is converted into an siRNA having a correct structure by the siRNA processing enzyme (Dicer or Rnase Ⅲ) existing in the cell, and the silencing of the target gene Guidedness is widely known.

As used herein, the term "antisense nucleic acid" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and is capable of binding to a complementary sequence in the mRNA, There are features that inhibit. The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to DJ-1 mRNA and capable of binding to DJ-1 mRNA and can be used for translation, translocation, maturation, And may inhibit essential activities for all other overall biological functions. The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. In the case of antisense RNA, it can be synthesized in vitro in a conventional manner and administered in vivo, or the antisense RNA can be synthesized in vivo. One example of the synthesis of antisense RNA in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector in which the origin of the recognition site (MCS) is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

As used herein, the term "inhibitor of activity of a DJ-1 protein" is a substance that inhibits the activity of a protein level of DJ-1, preferably an antibody or a compound that specifically binds to DJ-1.

The term "antibody" as used herein includes both monoclonal antibodies and chimeric, humanized and human antibodies thereto, and may include antibodies already known in the art in addition to the novel antibodies. The antibody comprises a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains, so long as it has the property of binding specifically recognizing DJ-1. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv. In addition, the compound which inhibits the activity of DJ-1 may be used without limitation as long as it plays a role of inhibiting the activity of DJ-1 in a hypoxic state of cancer cells. It may act directly on DJ-1, Or a compound which inhibits the activity by interfering with the binding with the ligand.

As used herein, the term "breast cancer" includes primary breast cancer, a malignant tumor that occurs primarily in the breast, or metastatic breast cancer that occurs when metastasis to other organs occurs in the breast. For example, in the case of ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), aqueous carcinoma, invasive lobular carcinoma (ILC), ductal carcinoma, mucinous carcinoma, but are not limited to, inflammatory breast cancer (IBC), lobular carcinoma in situ (LCIS), male breast cancer, Paget's disease or breast tumors of the breast.

The term "treatment ", as used herein, refers to the amelioration of one or more symptoms of breast cancer or the inhibition of the progression of symptoms. For example, in the context of the treatment of breast cancer, preference is given to reducing the rate of tumor growth, decreasing tumor size, decreasing the number of metastases, increasing tumor progression time, .

As used herein, the term "prophylactic" refers to a reduction in the incidence of pathological cells (e.g., hyperproliferative or neoplastic cells) in an animal. Prevention can be complete, for example, when there are no pathological cells in the subject at all. Prevention may also be partial, in which case the occurrence of pathologic cells within an individual means less than occurs when a pharmaceutical composition according to the invention is not used.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier. For oral administration, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersant, a stabilizer, a suspending agent, a colorant and a flavoring agent may be used. An isotonic agent, an isotonic agent, a stabilizer, etc. may be used in combination. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used.

The formulation of the pharmaceutical composition according to the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration. In the case of injections, it can be prepared in unit dosage ampoules or in multiple dosage forms.

The pharmaceutical composition according to the present invention can be administered via any conventional route so long as it can reach the target tissue. Intravenous, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intradermal, no.

The effective dose of the pharmaceutical composition according to the present invention can be varied depending on the sex, body surface area, kind and severity of disease, age, sensitivity to the drug, administration route and rate of release, administration time, treatment period, target cell, Other factors well known in the medical arts, and can be readily determined by one of ordinary skill in the art.

III. Methods of screening for preventive or therapeutic agents for breast cancer

The present invention

(a) treating the test substance with an expression cell line;

(b) measuring the level of expression or activity of the DJ-1 protein in the cell line; And

(c) selecting a test substance whose expression or activity level of the DJ-1 protein is lower than that of a control group in which the test substance is not treated, to screen for a preventive or therapeutic agent for breast cancer.

In step (b), the expression of the DJ-1 protein may be detected by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blotting, Assay (FACS), but are not limited thereto.

The activity of the DJ-1 protein in the step (b) can be measured by SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry or protein chip, but is not limited thereto.

Hereinafter, the present invention will be described in more detail by way of examples. However, the embodiments of the present invention are provided to facilitate the understanding of the invention, and the scope of protection of the invention is not limited by the following examples.

<Examples>

Example  1: Cell culture and transfection

Human fetal kidney cells 293 (HEK293), MCF7, MDA-MB-468 and SK-BR-3 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Dulbecco's Modified Eagle's Medium). MDA-MB-231 was cultured in RPMI supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. MCF10A cells were cultured in DMEM: F12 medium (Hyclone) supplemented with 5% horse serum, 100 ng / ml cholera toxin, 20 ng / ml EGF, 500 ng / ml hydrocortisone and 10 μg / ml insulin Lt; / RTI &gt; For plasmid DNA transfection, the cells were cultured at a density of 50-60%, grown overnight, and then transformed into an expression vector in the presence of plasmid DNA using calcium phosphate method or Lipofectamine-PLUS reagent.

Example  2: shRNA  Transfection

The pSUPER-derived expression vector for short hairpin RNA for Notch1 was prepared according to a conventional method. Mo et al., 2013, Notch1 modulates oxidative stress induced cell death and suppression of apoptosis signal-regulating kinase 1. Proceedings of the National Academy of Sciences Sciences of the United States of America 110; 6865-6870).

The following shRNA target sequences were used:

Notch1: 5'-AAGTGTCTGAGGCCAGCAAGA-3 '

Fbw7: 5'-CCTTCTCTGGAGAGAGAAA-3 '

Pin1: 5'-GCCATTTGAAGACGCCTCG-3 '

The specificity of the sequence was determined using a BLAST search and using a scrambled shRNA that did not match any known mammalian GENEBANK sequence as a control:

Scrambled shRNA: 5'-TTCTCCGAACGTGTCACGT-3 '

The DJ-1 target shRNA sequence is as follows:

DJ-1: 5'-UGGAGACGGUCAUCCCUGUdTdT-3 '(upper strand) and 3'-dTdTACCUCUGCCAGUAGGGACA-5' (lower strand).

The control and DJ-1 shRNA were transfected into cells using Lipofectamine-PLUS (Invitrogen) according to the manufacturer's instructions.

Example  3: Antibody

The blotted proteins were incubated with anti-Myc (9E10), anti-HA (12CA5), anti-Flag M2 (Sigma Chemical Co.), anti-DJ- Mouse HRP (horseradish peroxidase), and anti-mouse HRP (EMD Millipore), anti-Notch1, anti-RBP-Jk, anti-SMRT, anti-Fbw7, anti- And incubated with the conjugated secondary antibody (Amersham Biosciences, Inc.).

Example  4: RNA  Extraction and RT - PCR  And Q- PCR

Total RNA was extracted with Trizol reagent (Invitrogen, Camarillo, CA, USA), and the mixture was incubated with RNase-free DNase I (Takara, Toyko, Japan) at 37 ° C for 30 minutes under the following conditions: 20 mg of RNA, 40 mM, MgCl ₂ 8 mM, DTT 5 mM, RNase-inhibitor 0.4 U / mL (Promega, Madison, WI, USA) and 10 units of DNase I to a total volume of 50 mL. Then, RNA was precipitated using a phenol / chloroform extraction method. CDNA was synthesized with M-MLV reverse transcriptase (Promega) using total RNA (2 mg) according to the manufacturer's instructions. RNase-free DNase treatment was applied to remove the contaminating DNA. GAPDH was used as a control for PCR analysis. The primers used were as follows:

Primer set Hes1 (Forward) 5'- GGT GCT GAT AAC AGC GGA AT -3 ' (Reverse) 5'-TGA GCA AGT GCT GAG GGT TT -3 ' Hes5 (Forward) 5'-GGT GCC TCC ACT ATG ATC CTTA -3 ' (Reverse) 5'- TCC ACG TGA CTG AGT GTT CAA T -3 ' Hes6 (Forward) 5'- CGA AGT GCT GGA GCT GAC GGT G -3 ' (Reverse) 5'-CAC TGG ATG TAG CCG GCA GCG AA -3 ' Hey1 (Forward) 5'-GCA TCT CAA CAA CTA CGC TTC CCA -3 ' (Reverse) 5'- TGT GCG GGT GAT GTC CGAA -3 ' Hey2 (Forward) 5'-GGG TAA AGG CTA CTT TGA CGC AC-3 ' (Reverse) 5'-CGG AAT CCT ATG CTC ATG AA -3 ' p21 (Forward) 5'- GGC CCA GTG GAC AGC GAGCA -3 ' (Reverse) 5'-CCC AGG CGA AGT CAC CCT CC -3 ' p27 (Forward) 5'- GGT TAG CGG AGC AAT GCG-3 ' (Reverse) 5'- TCC ACA GAA CCG GCA TTT G -3 ' c-Myc (Forward) 5'- CTG TGG AAA AGA GGC AGG CT -3 ' (Reverse) 5'- GCT GTG AGG AGG TTT GCT GTG -3

Example  5: Immunoblotting  Test method

After 48 hours of transfection, the cultured HEK293 cells were harvested and cultured for 30 minutes at 4 ° C with RIPA (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 % SDS, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT and 2 ug each of leupeptin and aprotinin]. The disrupted cells were centrifuged at 12,000 g for 20 minutes at 4 ° C, and the resulting soluble fractions were denatured in Laemmli buffer and subjected to SDS-PAGE. After gel electrophoresis, the separated proteins were transferred to a PVDF (polyvinylidenedifluoride) membrane using electroblotting. The membrane was blocked with a Tris-buffered saline solution (pH 7) containing 0.1% Tween 20 and 5% skim milk. Blotting was performed using ECL (enhanced chemiluminescence).

Example  6: Luciferase  Reporter assay

Cells were lysed using a chemiluminescent lysis buffer containing 1 M K ₂ HPO ₄ , 18.3% 1 M KH ₂ PO ₄ , 1.7% PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT, and luciferase assay kit Promega) was used to analyze the luciferase activity. The activity of the luciferase reporter protein in the transfected cells was measured based on the? -Galactosidase activity measured in the same sample.

Example  7: Immunoprecipitation assays ( Co - immunoprecipitation Assay )

Cells were lysed with RIPA buffer at 4 &lt; 0 &gt; C for 30 minutes. After centrifugation at 12,000 g for 20 minutes, the supernatant was immunoprecipitated into protein A-agarose beads conjugated with the appropriate antibody. The resulting immunoprecipitates were washed three times with PBS (phosphate-buffered solution) at pH 7.4 and Laemmli sample buffer was added to the immunoprecipitation pellet. The pellet was heated at 95 DEG C for 5 minutes and subjected to SDS-PAGE analysis. Western blotting was performed using the antibody.

Example  8: In vitro  Binding assay

The recombinant His-DJ-1 protein was transformed into Escherichia It was expressed in BL21 strain coli. The DJ-1 protein was then purified using nickel beads (Sigma) according to the manufacturer's instructions. Equal amounts of His or His-DJ-1 fusion protein were incubated with lysates of HEK293 cells and transfected with the expression vector at 4 ° C for 3 hours with agitation. After incubation, the beads were washed three times with cold PBS and heated with 20 [mu] l Laemmli sample buffer. The precipitate was separated by SDS-PAGE and the pull-down protein was detected by immunoblotting with a specific antibody.

Example  9: Protein stability test

Cells were seeded at 50-60% density and cultured overnight. When the cells were added to the medium, they were treated with 0.1 mM cycloheximide for 0, 0.5, 1, 2, 4, or 6 hours to block synthesis with new proteins, collected for each hour, and analyzed using RIPA or Laemmli buffer Lt; / RTI &gt; The protein level of endogenous Notch1 was measured by immunoblotting using anti-Notchl antibody diluted 1: 3000.

Example  10: Immunofluorescent staining

Cells were fixed with PBS containing 4% paraformaldehyde, and PBS containing 0.1% Triton-X 100 dissolved therein was infiltrated. The cells were blocked with PBS supplemented with 1% BSA and reacted with primary antibody diluted 1: 100. After reacting with the primary antibody, the cells were washed three times with PBS, and an anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen) or Alexa Fluor 532 (Invitrogen) diluted 1: 100 was added And DNA was stained using TO-PRO®-3 Iodide. The stained cells were detected using a confocal microscope (Leica TCS SPE) and each image was observed in a single Z zone at the same cell level. The final images were analyzed using a confocal microscope equipped with LAS AF software (Leica).

Example  11: Wound healing  Test method

Cells were grown in 12-well plates and monolayers were wounded using a P200 micropipette tip. After the measurements, DMEM supplemented with 10% FBS was immediately replaced. Wound-healing images were taken at 6-hour intervals using a microscope system. The cell migration rate was calculated by measuring the distance of the sketched region at each point using the Image J program.

Example  12: Colony formation  And soft agar assay

The colony formation assay was performed by seeding 500 cells in a cell-growth matrix consisting of bottom agar and top agarose in a 6 well culture plate. The lower layer (1.5 ml) contained DMEM medium, 10% FBS and 0.5% agar, and the upper layer (1.5 ml) contained DMEM medium, 10% FBS, 0.35% agarose and cell suspension (1 x 10 ³ ). Cells were fed every 3 days and colonies were measured 2-3 weeks later.

Example  13: Cell migration and invasion assay

Transwell migration assays were performed using a transwell chamber (8 [mu] m polycarbane membrane, Corning). Matrigel cell invasion assay was performed with BD BioCoat Growth Factor. Typically, 2.5 x 10 ⁴ to 2.5 x 10 ⁵ cells were seeded into a chamber coated with 10 μg / ml fibronectin. 37 ° C, 5% CO ₂ For 16 hours, and the cells attached to the upper surface of the filter were removed with a cotton swab. Cells were fixed with 3.7% formaldehyde or 100% methanol and stained with DAPI to determine the number of cells. The number of migrated cells was determined by the number of stained cells in a randomly selected multiple microscopic field of view.

Example  14: Mouse transplantation and transfer model

Female C57BL / 6 mice (Orient Bio, Korea) were maintained in an aseptic facility in an animal laboratory at the National Cancer Center. MDA-MB-231-SQ breast cancer cell lines (1 x 10 ⁶ cells / ml in 100 μl PBS) stably expressing specific shRNAs for shCon (control), DJ-1 and Fbs7 were transfected into 6-week-old female C57BL / 5 &lt; / RTI &gt; per mouse) in the tail vein. After 4 weeks, the mice were euthanized via anesthetic overdose. Tumor size was measured with a caliper and the volume of the dose was calculated according to the formula V = (L x W ² ) x 0.5 L: length of the tumor, W: width of the tumor).

Statistical processing

Data were expressed as mean ± standard deviation of three replicate experiments, and statistical significance was measured by two-tailed paired Student's t-test. ** P <0.01 was considered statistically significant.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> A PHARMACEUTICAL COMPOSITION FOR TREATING OR PREVENTING BREAST          CANCER COMPRISING DJ-1 INHIBITOR AS AN ACTIVE INGREDIENT <130> DPP-2014-0247 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> DJ-1 <400> 1 Met Ala Ser Lys Arg Ala Leu Val Ile Leu Ala Lys Gly Ala Glu Glu   1 5 10 15 Met Glu Thr Val Ile Pro Val Asp Val Met Arg Arg Ala Gly Ile Lys              20 25 30 Val Thr Val Ala Gly Leu Ala Gly Lys Asp Pro Val Gln Cys Ser Arg          35 40 45 Asp Val Valle Cys Pro Asp Ala Ser Leu Glu Asp Ala Lys Lys Glu      50 55 60 Gly Pro Tyr Asp Val Val Val Leu Pro Gly Gly Asn Leu Gly Ala Gln  65 70 75 80 Asn Leu Ser Glu Ser Ala Ala Val Lys Glu Ile Leu Lys Glu Gln Glu                  85 90 95 Asn Arg Lys Gly Leu Ile Ala Ala Ile Cys Ala Gly Pro Thr Ala Leu             100 105 110 Leu Ala His Glu Ile Gly Phe Gly Ser Lys Val Thr Thr His Pro Leu         115 120 125 Ala Lys Asp Lys Met Met Asn Gly Gly His Tyr Thr Tyr Ser Glu Asn     130 135 140 Arg Val Glu Lys Asp Gly Leu Ile Leu Thr Ser Arg Gly Pro Gly Thr 145 150 155 160 Ser Phe Glu Phe Ala Leu Ala Ile Val Glu Ala Leu Asn Gly Lys Glu                 165 170 175 Val Ala Gln Val Lys Ala Pro Leu Val Leu Lys Asp             180 185

Claims (13)

delete delete delete As a method for suppressing the expression of the DJ-1 gene, which comprises blocking the signaling pathway of Notch1 in an animal other than a human,
Wherein the DJ-1 comprises the amino acid sequence of SEQ ID NO: 1,
The Notch1 signaling pathway block
Notch1, or the Notch1 target gene comprising Hes1 and Hes5,
Down-regulation of the interaction of Notch1 with RBP-Jk, or
RTI ID = 0.0 &gt; DJ-1 &lt; / RTI &gt; gene, wherein the interaction of Notch1 and Fbw7 is upregulated. delete delete delete delete delete delete delete delete delete

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